Generation of HPV-specific T-cells

ABSTRACT

Embodiments of the disclosure concern methods and compositions for immunotherapy for human papillomavirus infection and diseases associated therewith. In specific embodiments, methods concern production of immune cells that target one or more antigens of HPV16 and/or HPV18, including methods with stimulation steps that employ IL-7 and IL-15, but not IL-6 and/or IL-12. Other specific embodiments utilize stimulations in the presence of certain cells, such as costimulatory cells and certain antigen presenting cells.

RELATED APPLICATIONS

This application is a continuation-in-part application of InternationalApplication Serial No. PCT/EP2017/073274, filed 15 Sep. 2017.International Application Serial No. PCT/EP2017/073274 claims priorityto U.S. Provisional Patent Application Ser. No. 62/395,440, filed 16Sep. 2016 and U.S. Utility application Ser. No. 15/331,659, filed 21Oct. 2016. U.S. Utility application Ser. No. 15/331,659 also claimspriority to U.S. Provisional Patent Application Ser. No. 62/395,440.This application also claims priority to International ApplicationSerial No. PCT/US17/51284, filed 13 Sep. 2017. International ApplicationSerial No. PCT/US17/51284 claims priority to U.S. Provisional PatentApplication Ser. No. 62/395,438, filed 16 Sep. 2016. All of thereferenced applications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under P50 CA097007 andPO1 CA94237 awarded by National Cancer Institute. The government hascertain rights in the invention.

TECHNICAL FIELD

The present disclosure concerns at least the fields of immunology, cellbiology, molecular biology, and medicine, including cancer medicine.

BACKGROUND

Human papillomavirus (HPV) is a DNA virus that establishes productiveinfections in keratinocytes of the skin or mucous membranes. There areover 170 types of HPV, a subset of which HPV types are carcinogenic,including high-risk sexually transmitted types that can develop intogenital neoplasias, including cervical intraepithelial neoplasia (CIN),vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia(PIN), and/or anal intraepithelial neoplasia (AIN), for example.HPV—induced cancers arise when viral sequences are integrated into thecellular DNA of host cells. Some of the HPV “early” genes, such as E6and E7, act as oncogenes that promote tumor growth and malignanttransformation.

Ramos et al., (J Immunother 2013; 36:66-76) describes a method forstimulating peripheral blood mononuclear cells to generate T-cellsspecific for HPV16 E6 and E7. In brief, the method comprises stimulationof peripheral blood mononuclear cells with dendritic cells in whichcells are cultured in CTL medium with or without the combination ofcytokines IL-6, IL-7, IL-12 and IL-15, a second stimulation in whichco-cultures are supplemented with IL-2, and weekly stimulation withpepmix-loaded accessory antigen presenting cells (e.g., B-blasts) in thepresence of IL-15. This reference teaches the combination of cytokinesIL-6, IL-7, IL-12 and IL-15 is required for expansion of theHPV-specific T-cells from patient samples, for detectable T-cellresponses.

The present disclosure provides relief for a long-felt need in the artto treat HPV-associated diseases, including at least for thoseassociated with HPV16 and HPV18, for example.

BRIEF SUMMARY

The present disclosure is directed to methods and compositions thatconcern immune system cells that are modified to immunogenicallyrecognize particular targets. In some embodiments, the presentdisclosure concerns the development of immune cells (including cytotoxicT-lymphocytes (CTLs, also referred to as cytotoxic T-cells)) that targeta biological moiety that elicits an immune response in an individual. Inspecific embodiments, the present disclosure concerns the development ofcytotoxic T-cells that target a HPV antigen, including a HPVdisease-associated antigen. In some cases, a mixture of cytotoxicT-cells is produced, and the mixture targets more than one HPV antigen,including more than one antigen of more than one HPV type, in somecases.

Embodiments of the disclosure concern methods and compositions forproviding therapy to individuals infected with HPV or that haveHPV-associated diseases, including cancers, for example. A“HPV-associated disease” may be a disease which is caused or exacerbatedby HPV infection, a disease for which HPV infection is a risk factorand/or a disease for which HPV infection is positively associated withdisease onset, development, progression or severity. A HPV-associateddisease may be a disease in which the methods and compositions of thepresent invention provide therapeutic effect (e.g. inhibition of thedevelopment/progression of the disease, delayed/prevented onset of thedisease, reduced severity of the symptoms of the disease, reversal ofdisease symptoms, and/or increased survival). It will be clear to theperson skilled in the art that the therapeutic utility of the methodsand compositions of the present invention extends to essentially anydisease/condition which would benefit from a reduction in the number ofHPV-infected cells. In specific embodiments, the disclosure regardsmethods and compositions for adoptive cellular immunotherapy that cantarget HPV-associated, e.g., HPV16-associated, HPV18-associated,HPV1-associated, HPV2-associated and/or HPV3-associated medicalconditions (including cancer) and are therapeutic therefor.

In certain aspects, the present disclosure concerns the development of aplurality of T-cells that target antigens from HPV, e.g., HPV16, HPV18,HPV1, HPV2 and/or HPV3. The present disclosure provides significant andnon-obvious improvements on methods for generating T cell lines withspecificity against HPV, e.g., HPV16, HPV18, HPV1, HPV2 and/or HPV3antigens.

In some embodiments of the disclosure, an individual is in need of themethods and/or compositions of the disclosure. In certain embodiments,an individual has been exposed to HPV, e.g., HPV16, HPV18, HPV1, HPV2and/or HPV3 (the presence of which may or may not be known for theindividual), or the individual is suspected of having been exposed to orat risk for being exposed to HPV, e.g., HPV16, HPV18, HPV1, HPV2 and/orHPV3. In certain embodiments, the individual has or is suspected ofhaving or is at risk for having HPV-associated disease, e.g.,HPV16-associated, HPV18-associated, HPV1-associated, HPV2-associatedand/or HPV3-associated disease, including cancer.

In specific embodiments of part of the method, certain HPV, e.g., HPV16,HPV18, HPV1, HPV2 and/or HPV3, antigen(s) are presented toantigen-presenting cells (APCs) in the form of one or more peptides thatspan some or all of certain antigen(s). The antigenic peptides may beprovided to the antigen-presenting cells in a library of peptidemixtures, which may be referred to as pepmixes. In certain aspects ofthe disclosure, there is pooling of a variety of pepmixes for exposureto the APCs. APCs that express the antigens may be exposed to peripheralblood T-cells under certain conditions to result in stimulation ofT-cells specific for the certain HPV antigen(s).

Some aspects and embodiments of the present disclosure concern thegeneration and/or expansion of HPV-specific T-cells.

In a first aspect, the present disclosure provides a method forstimulating peripheral blood cells, preferably peripheral blood T-cells,wherein the method comprises stimulating peripheral blood T-cells withantigen presenting cells in the presence of interleukin (IL)-7 and IL-15and, in at least some cases, in the absence of IL-6 and/or IL-12,wherein the antigen presenting cells were previously exposed to one ormore peptides, wherein the peptides comprise sequence that correspondsto at least part of the sequence of one or more proteins of HPV. Inaccordance with various aspects disclosed herein, where astimulation/culture is performed in the “presence of” a given cytokine,the relevant cytokine (e.g. recombinant and/or exogenous cytokine) mayhave been added to the stimulation/culture. Where a stimulation/cultureis performed in the “absence of” a given cytokine, the relevant cytokine(e.g. recombinant and/or exogenous cytokine) will not have been added tothe stimulation/culture.

In some embodiments a method of producing therapeutic T-cells for humanpapillomavirus (HPV)-associated disease(s) is provided, the methodcomprising the step of stimulating peripheral blood T-cells with antigenpresenting cells in the presence of one or more of interleukin IL-7 andIL-15 and, in at least some cases, in the absence of IL-6 and/or IL-12,wherein the antigen presenting cells were previously exposed to one ormore peptides, wherein the peptides comprise sequence that correspondsto at least part of the sequence of one or more proteins of HPV, whereinthe stimulating produces T-cells therapeutic for HPV-associateddiseases.

In some embodiments, the peripheral blood T-cells being stimulated areobtained from a prior stimulation of peripheral blood cells. The priorstimulation may comprise stimulating peripheral blood cells with antigenpresenting cells in the presence of IL-7 and IL-15, and in at least somecases in the presence of IL-6 and/or IL-12, wherein the antigenpresenting cells were previously exposed to one or more peptides,wherein the peptides comprise sequence that corresponds to at least partof the sequence of one or more proteins of HPV.

As such, prior to stimulating the peripheral blood T-cells, the methodsmay further comprise stimulating peripheral blood cells with antigenpresenting cells in the presence of IL-7 and IL-15, and in at least somecases in the presence of IL-6 and/or IL-12, wherein the antigenpresenting cells were previously exposed to one or more peptides,wherein the peptides comprise sequence that corresponds to at least partof the sequence of one or more proteins of HPV, to produce peripheralblood T-cells.

In some embodiments the one or more peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV16; one or more proteins of HPV18; or both of one or more proteins ofHPV16 and one or more proteins of HPV18. In some embodiments the one ormore peptides comprise sequence that corresponds to at least part of thesequence of one or more proteins of HPV1; one or more proteins of HPV2;one or more proteins of HPV3; or one or more proteins of HPV1, HPV2and/or HPV3. In some embodiments the one or more peptides comprisesequence that corresponds to one or more of proteins E5, E6, E7, L1 andL2. In some embodiments the one or more peptides may be a library ofpeptides, including E1, E2, E3, E4, E5, E6, E7, L1, and/or L2 peptides.

In some embodiments the method may produce immune cells, such asT-cells, specific for HPV or for an HPV antigen. In some embodiments themethod may expand a population of T-cells present in the peripheralblood T-cells that are specific for HPV or for at least one HPV antigenImmune cells other than T cells that may be produced by methods of thedisclosure including NK cells and NKT cells.

In some embodiments the antigen presenting cells are activated T-cells,dendritic cells (DC), B-Blasts (BB), or PBMCs, for example.

In some embodiments stimulation of peripheral blood T-cells in thepresence of IL-7 and IL-15 occurs in the absence of at least IL-2. Insome embodiments stimulation of peripheral blood T-cells in the presenceof IL-7 and IL-15 occurs in the absence of at least IL-4. In someembodiments stimulation of peripheral blood T-cells in the presence ofIL-7 and IL-15 occurs in the absence of at least IL-6. In someembodiments stimulation of peripheral blood T-cells in the presence ofIL-7 and IL-15 occurs in the absence of at least IL-12. In someembodiments stimulation of peripheral blood T-cells in the presence ofIL-7 and IL-15 occurs in the absence of at least IL-21. In someembodiments stimulation of peripheral blood T-cells in the presence ofIL-7 and IL-15 occurs in the absence of IL-6 and IL-12.

In some particular embodiments stimulation of cells in the method of thefirst aspect of the present invention occurs in the absence of IL-6 andIL-12.

In some embodiments, peripheral blood T-cells may be present in apopulation of peripheral blood mononuclear cells (PBMCs) or are obtainedor isolated therefrom. The PBMCs in the population may be non-adherentPBMCs. The antigen presenting cells may be activated T-cells, dendriticcells, B-blasts, or PBMCs, for example.

In a second aspect, the present disclosure provides a method forstimulating T-cells specific for HPV or for an HPV antigen, wherein themethod comprises stimulating T-cells specific for HPV or for an HPVantigen with antigen presenting cells in the presence of IL-7 and IL-15,and optionally in the presence of co-stimulatory cells, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV.

In some embodiments a method of producing therapeutic T-cells for humanpapillomavirus (HPV)-associated diseases is provided, the methodcomprising the step of stimulating T-cells specific for HPV or for anHPV antigen with antigen presenting cells in the presence of one or moreof interleukin IL-7 and IL-15, and optionally in the presence ofco-stimulatory cells, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV, wherein the stimulating produces T-cellstherapeutic for one or more HPV-associated diseases.

In some embodiments the antigen presenting cells are activated T-cells,dendritic cells (DC), B-Blasts (BB) or PBMCs. In particular embodimentsthe antigen presenting cells are activated T-cells.

In some embodiments the co-stimulatory cells are one or more cell typesselected from the group consisting of CD80+ cells, CD86+ cells, CD83+cells, 4-1BBL+ cells, CD40+ cells, OX40+ cells, and a combinationthereof. The co-stimulatory cells may be CD80+/CD86+/CD83+/4-1BBL+cells.

In some embodiments the stimulation of T-cells specific for HPV or foran HPV antigen is not a first stimulation step. The T-cells beingstimulated cells may be the product of a prior stimulation, e.g. usingthe method of the first aspect of the present disclosure.

In some embodiments the one or more peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV16; one or more proteins of HPV18; or one or more proteins of HPV16and one or more proteins of HPV18. In some embodiments the one or morepeptides comprise sequence that corresponds to at least part of thesequence of one or more proteins of HPV1; one or more proteins of HPV2;one or more proteins of HPV3; or one or more proteins of HPV1, HPV2and/or HPV3. In some embodiments the one or more peptides comprisesequence that corresponds to one or more of proteins E5, E6, E7, L1 andL2. In some embodiments the one or more peptides may be a library ofpeptides, including E1, E2, E3, E4, ES, E6, E7, L1, and/or L2 peptides.

In some embodiments the method may produce T-cells specific for HPV orfor an HPV antigen. In some embodiments the method may expand apopulation of T-cells specific for HPV or for an HPV antigen.

In certain embodiments stimulation of T-cells specific for HPV or for anHPV antigen comprises stimulating T-cells specific for HPV or for an HPVantigen with antigen presenting cells in the presence of IL-7, IL-15,and in the presence of one or more types of co-stimulatory cells.

In some embodiments stimulation of T-cells in the presence of IL-7 andIL-15 is in the absence of IL-2. In some embodiments stimulation ofT-cells in the presence of IL-7 and IL-15 is in the absence of IL-4. Insome embodiments stimulation of T-cells in the presence of IL-7 andIL-15 is in the absence of IL-6. In some embodiments stimulation ofT-cells in the presence of IL-7 and IL-15 is in the absence of IL-7. Insome embodiments stimulation of T-cells in the presence of IL-7 andIL-15 is in the absence of IL-12. In some embodiments stimulation ofT-cells in the presence of IL-7 and IL-15 is in the absence of IL-21. Insome embodiments stimulation of T-cells in the method of the firstaspect of the present invention is in the absence of IL-6 and IL-12.

Methods according to the first and second aspect of the presentdisclosure may be methods of producing therapeutic T-cells forHPV-associated diseases. The stimulation of cells may produce T-cellsthat are therapeutic for HPV-associated diseases.

In a third aspect, the methods of the first and second aspects may becombined to provide a method of producing therapeutic T-cells forHPV-associated diseases, the method comprising:

stimulating peripheral blood cells, preferably peripheral blood T-cells,wherein the method comprises stimulating peripheral blood T-cells withantigen presenting cells in the presence of interleukin (IL)-7 andIL-15, and optionally in the absence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV;

stimulating T-cells obtained from (i) with antigen presenting cells inthe presence of interleukin (IL)-7 and IL-15, and optionally in thepresence of one or more types of co-stimulatory cells, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV.

In some embodiments prior to step (ii), T-cells obtained from (i) may bere-stimulated in the presence of IL-7 and IL-15 but not in the presenceof co-stimulatory cells, and optionally in the absence of IL-6 and/orIL-12. Such re-stimulation may occur for one, two, three, four, five ormore rounds, as required.

In some embodiments the antigen presenting cells used in (i) aredendritic cells (DC), B-Blasts (BB) or PBMCs. In some embodiments theantigen presenting cells used in (ii) are activated T-cells, dendriticcells (DC), B-Blasts (BB) or PBMCs. In some embodiments the antigenpresenting cells used in (i) are different to the antigen presentingcells used in (ii), although they may be the same in certain cases. Inparticular embodiments the antigen presenting cells used in (ii) areactivated T-cells.

In some embodiments the co-stimulatory cells are one or more cell typesselected from the group consisting of CD80+ cells, CD86+ cells, CD83+cells, 4-1BBL+ cells, CD40+ cells, OX40+ cells, and a combinationthereof. The co-stimulatory cells may be CD80+/CD86+/CD83+/4-1BBL+cells.

In some embodiments stimulation of cells in the presence of IL-7 andIL-15 is in the absence of IL-2. In some embodiments stimulation ofcells in the presence of IL-7 and IL-15 is in the absence of IL-4. Insome embodiments stimulation of cells in the presence of IL-7 and IL-15is in the absence of IL-6. In some embodiments stimulation of cells inthe presence of IL-7 and IL-15 is in the absence of IL-12. In someembodiments stimulation of cells in the presence of IL-7 and IL-15 is inthe absence of IL-21. In some embodiments stimulation of cells in thepresence of IL-7 and IL-15 is in the absence of IL-6 and IL-12.

In some preferred embodiments stimulation of cells in step (i) is in theabsence of IL-6 and IL-12.

In some embodiments stimulation of cells in step (ii) is in the absenceof IL-6 and IL-12.

Accordingly, in some embodiments a method of producing therapeuticT-cells for HPV-associated diseases is provided, the method comprising:

(i) stimulating peripheral blood cells, wherein the method comprisesstimulating peripheral blood T-cells with antigen presenting cells inthe presence of interleukin (IL)-7 and IL-15 and optionally in theabsence of IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV;

(ii) stimulating T-cells obtained from (i) with antigen presenting cellsin the presence of interleukin (IL)-7 and IL-15 and optionally in theabsence of IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV, wherein (ii) is optionally repeated one ormore times; and

(iii) stimulating T-cells obtained from (ii) with antigen presentingcells in the presence of IL-7 and IL-15, and optionally in the presenceof co-stimulatory cells, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV, wherein (iii) is optionally repeated one ormore times.

In some embodiments the antigen presenting cells used in (i) and (ii)are dendritic cells (DC) B-Blasts (BB) or PBMCs. In some embodiments theantigen presenting cells used in (iii) are activated T-cells, dendriticcells (DC), B-Blasts (BB) or PBMCs. In some embodiments the antigenpresenting cells used in (iii) are different to the antigen presentingcells used in (i) and/or (ii). In preferred embodiments the antigenpresenting cells used in (iii) are activated T-cells.

In preferred embodiments the stimulation in (iii) is in the presence ofco-stimulatory cells. In some embodiments the co-stimulatory cells areone or more cell types selected from the group consisting of CD80+cells, CD86+ cells, CD83+ cells, 4-1BBL+ cells, CD40+ cells, OX40+cells, and a combination thereof. The co-stimulatory cells may beCD80+/CD86+/CD83+/4-1BBL+ cells.

In some embodiments stimulation of cells in the presence of IL-7 andIL-15 is in the absence of IL-2. In some embodiments stimulation ofcells in the presence of IL-7 and IL-15 is in the absence of IL-4. Insome embodiments stimulation of cells in the presence of IL-7 and IL-15is in the absence of IL-6. In some embodiments stimulation of cells inthe presence of IL-7 and IL-15 is in the absence of IL-12. In someembodiments stimulation of cells in the presence of IL-7 and IL-15 is inthe absence of IL-21. In some embodiments stimulation of cells in thepresence of IL-7 and IL-15 is in the absence of IL-6 and IL-12.

In some embodiments stimulation of cells in step (i) is in the absenceof IL-6 and IL-12. In some other embodiments stimulation of cells instep (i) is in the presence of IL-6 and IL-12.

In some particular embodiments stimulation of cells in step (ii) is inthe absence of IL-6 and IL-12. In some particular embodimentsstimulation of cells in step (iii) is in the absence of IL-6 and IL-12.

In some particular embodiments methods of the present disclosure are forproducing T-cells specific for HPV16 and/or HPV18. In some particularembodiments methods of the present invention are for producing T-cellsspecific for HPV16-associated and/or HPV18-associated diseases.

In some embodiments, peripheral blood T-cells may be obtained from anindividual that is known to be infected or suspected of being infectedwith HPV; HPV16 or HPV18; both HPV16 and HPV18; HPV1 , HPV2 and/or HPV3.

In some embodiments, antigen presenting cells may be obtained from anindividual that is known to be infected or suspected of being infectedwith HPV; HPV16 or HPV18; both HPV16 and HPV18; HPV1 , HPV2 and/or HPV3.

In some embodiments, the method may occur in the absence of exposing theT-cells produced by the method to activated B cells that were previouslyexposed to a library of peptides.

In some embodiments, antigen presenting cells may be autologous orallogeneic to an individual intended to be treated with the therapeuticT-cells obtained.

In some embodiments, the one or more peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV16; one or more proteins of HPV18; or one or more proteins of HPV16and one or more proteins of HPV18. In some embodiments the one or morepeptides comprise sequence that corresponds to at least part of thesequence of one or more proteins of HPV1; one or more proteins of HPV2;one or more proteins of HPV3; or one or more proteins of HPV1, HPV2and/or HPV3. In some embodiments the one or more peptides comprisesequence that corresponds to one or more of proteins E1, E2, E3, E4, E5,E6, E7, L1, and/or L2 that come from HPV16, HPV18, or HPV16 and HPV18.

In embodiments of the present disclosure the peptides may comprisesequence that corresponds to one or more of HPV proteins E1, E2, E3, E4,E5, E6, E7, L1, and/or L2. In some embodiments, the peptides maycomprise sequence that corresponds to one or more HPV proteins which areexpressed following proviral integration (e.g. E1, E2, E3, E4, E5, E6and/or E7), e.g. in a cell infected HPV. In some embodiments, thepeptides may comprise sequence that corresponds to one or moretransforming HPV proteins (e.g. E6, and/or E7). The peptides maycorrespond to a contiguous amino acid sequence present within said HPVprotein. A peptide may have a length of at least or no more than 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length, orof 15 amino acids in length. The collection of peptides may form alibrary and peptides in the library may overlap in sequence with otherpeptides by any suitable amount, including 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or 14 amino acids, for example. The peptides may comprisesequence that corresponds to: a) the HPV18 E6 protein and/or the HPV18E7 protein, and/or b) the HPV16 E6 protein and/or the HPV16 E7 protein.

In embodiments of the present disclosure the HPV may be HPV16 or HPV18,or both. In embodiments concerned with treatment of an HPV-associateddisease, the disease may be cancer and the peptides may comprise asequence that corresponds to one or both of E6 and E7. When theHPV-associated disease is a pre-cancerous lesion, the peptides maycomprise sequence that corresponds to one, some, or all of E1, E2, E3,E4, E5, E6, E7, L1, and L2.

T-cells produced by the methods of the present disclosure may beisolated and/or purified, e.g., isolated/purified from other cells.

In some embodiments, a therapeutically effective amount of T-cellsproduced by the methods of the present disclosure are provided to anindividual that has been exposed to HPV, or that has HPV-associateddisease. In a related aspect T-cells produced by the method of thepresent disclosure are provided for use in the treatment ofHPV-associated disease. In another related aspect the use of T-cellsproduced by the method of the present disclosure are provided for use inthe manufacture of a medicament for use in the treatment ofHPV-associated disease.

In one aspect of the present invention T-cells for use in a method ofadoptive cellular immunotherapy are provided, wherein the T-cells areobtained by, obtainable by, or are the product of, a method forstimulating peripheral blood or T-cells or a method of producingtherapeutic T-cells described herein, the method of adoptive cellularimmunotherapy comprising administering the T-cells to the subject.

In one aspect of the present invention the use of T-cells in themanufacture of a medicament for use in a method of adoptive cellularimmunotherapy comprising administering the T-cells to the subject isprovided, wherein the T-cells are obtained by, obtainable by, or are theproduct of, a method for stimulating peripheral blood or T-cells or amethod of producing therapeutic T-cells described herein.

In one aspect of the present invention a method of preparing apharmaceutical composition, medicament or vaccine is provided, themethod comprising stimulating peripheral blood or T-cells according to amethod described herein, or producing therapeutic T-cells according to amethod described herein, and mixing the cells obtained, with apharmaceutically acceptable carrier, adjuvant, diluent or excipient.

The disease to be treated may be a neoplasm. The neoplasm may be acancer. The cancer may be an HPV-positive cancer, e.g. a HPV16-positivecancer and/or HPV18-positive cancer.

The individual to be treated may be a human. The individual may be apatient. The individual may have been exposed to HPV, such as HPV16,HPV18, or both HPV16 and HPV18, or has an HPV-, HPV16- and/orHPV18-associated disease. The HPV-, HPV16- and/or HPV18-associateddisease may be a neoplasm. The neoplasm may be a cancer.

A cancer may be of any kind. In some embodiments the cancer is acervical cancer, anal cancer, vulvar cancer, vaginal cancer, penilecancer, or oropharyngeal cancer. In some embodiments the cancer may be aHPV-associated cancer. A “HPV-associated cancer” may be a cancer whichis caused or exacerbated by HPV infection, a cancer for which HPVinfection is a risk factor and/or a cancer for which HPV-infection ispositively associated with onset, development, progression, severity ormetastasis. A HPV-associated cancer may be a cancer in which the methodsand compositions of the present invention provide therapeutic effect(e.g. inhibition of the development/progression of the cancer,delayed/prevented onset of the cancer, reduced/delayed/preventedmetastasis, reduced severity of the symptoms of the cancer, reduction innumber of cancer cells, reduction in tumour size, and/or increasedsurvival). In some embodiments the cancer is a HPV-related carcinoma,HPV-positive oropharyngeal carcinoma, HPV-positive cervical carcinoma,HPV-positive anal carcinoma, HPV-positive vulvar carcinoma,nasopharyngeal carcinoma, HPV-positive penile carcinoma, HPV-positivedysplasias of any site, or laryngeal papillomatosis.

The individual or subject may have received, be receiving, or willreceive an additional cancer therapy. The additional cancer therapy maybe surgery, radiation, hormone therapy, chemotherapy, immunotherapy, ora combination thereof.

The individual or subject may be determined as having HPV-associatedcancer or HPV-positive cancer. The individual may be determined ashaving HPV16-associated cancer or HPV16-positive cancer. The individualmay be determined as having HPV18-associated cancer or HPV18-positivecancer. The individual or subject may be any animal or human. Theindividual or subject is preferably mammalian, more preferably human.The individual or subject may be a non-human mammal, but is morepreferably human. The individual or subject may be male or female. Theindividual or subject may be a patient.

Methods according to the present disclosure that involve steps of cellstimulation may be performed in vitro or ex vivo. The term “in vitro” isintended to encompass studies with materials, biological substances,cells and/or tissues in laboratory conditions or in culture. “Ex vivo”refers to something present or taking place outside an organism, e.g.outside the human or animal body, which may be on tissue (e.g. wholeorgans) or cells taken from the organism.

In one embodiment, there is a method for stimulating peripheral bloodcells, the method comprising stimulating peripheral blood T-cells withantigen presenting cells in the presence of interleukin (IL)-7 and IL-15and in the absence of IL-6 and/or IL-12, wherein the antigen presentingcells were previously exposed to one or more peptides, wherein thepeptides comprise sequence that corresponds to at least part of thesequence of one or more proteins of human papillomavirus (HPV). Theperipheral blood T-cells may be obtained from a prior stimulation ofperipheral blood cells, such as wherein the prior stimulation ofperipheral blood cells comprises stimulating peripheral blood cells withantigen presenting cells in the presence of IL-7 and IL-15, and in thepresence of IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV. In specific cases, prior to stimulating theperipheral blood T-cells, the method further comprises stimulatingperipheral blood cells with antigen presenting cells in the presence ofIL-7 and IL-15, and in the presence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV, to produceperipheral blood T-cells.

In an embodiment, there is a method of producing therapeutic T-cells forHPV-associated diseases, the method comprising the step of: stimulatingperipheral blood T-cells with antigen presenting cells in the presenceof one or more of IL-7 and IL-15 and in the absence of IL-6 and/orIL-12, wherein the antigen presenting cells were previously exposed toone or more peptides, wherein the peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV, wherein the stimulating produces T-cells therapeutic forHPV-associated diseases. The peripheral blood T-cells may be obtainedfrom a prior stimulation of peripheral blood cells, such as wherein theprior stimulation of peripheral blood cells comprises stimulatingperipheral blood cells with antigen presenting cells in the presence ofIL-7 and IL-15, and in the presence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV. In specificcases, prior to stimulating the peripheral blood T-cells, the methodfurther comprises stimulating peripheral blood cells with antigenpresenting cells in the presence of IL-7 and IL-15, and in the presenceof IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV, to produce peripheral blood T-cells.

In embodiments of methods encompassed by the disclosure, antigenpresenting cells are activated T-cells, dendritic cells, B-blasts, orPBMCs. Peripheral blood T-cells may be present in a population ofperipheral blood mononuclear cells (PBMCs) or are obtained or isolatedtherefrom, in at least some cases, and the PBMCs in the population maybe non-adherent PBMCs. When employed, co-stimulatory cells may be CD80+,CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells, or a combinationthereof.

In a particular embodiment, there is a method for stimulating T-cellsspecific for HPV or for an HPV antigen, the method comprisingstimulating T-cells specific for HPV or for an HPV antigen with antigenpresenting cells in the presence of IL-7 and IL-15 and in the presenceof co-stimulatory cells, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV.

In certain embodiments, there is a method of producing therapeuticT-cells for HPV-associated diseases, the method comprising the step ofstimulating T-cells specific for HPV or for an HPV antigen with antigenpresenting cells in the presence of one or more of IL-7 and IL-15 and inthe presence of co-stimulatory cells, wherein the antigen presentingcells were previously exposed to one or more peptides, wherein thepeptides comprise sequence that corresponds to at least part of thesequence of one or more proteins of HPV, wherein the stimulatingproduces T-cells therapeutic for HPV-associated diseases.

In one embodiment, there is a method of producing therapeutic T-cellsfor HPV-associated diseases, the method comprising: (i) stimulatingperipheral blood cells, wherein the method comprises stimulatingperipheral blood T-cells with antigen presenting cells in the presenceof IL-7 and IL-15 and optionally in the absence of IL-6 and/or IL-12,wherein the antigen presenting cells were previously exposed to one ormore peptides, wherein the peptides comprise sequence that correspondsto at least part of the sequence of one or more proteins of HPV; and(ii) stimulating T-cells obtained from (i) with antigen presenting cellsin the presence of IL-7 and IL-15, and in the presence of co-stimulatorycells, wherein the antigen presenting cells were previously exposed toone or more peptides, wherein the peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV. In specific embodiments, prior to step (ii) T-cells obtained from(i) may be re-stimulated in the presence of IL-7 and IL-15 but not inthe presence of co-stimulatory cells.

In an embodiment, a method of producing therapeutic T-cells forHPV-associated diseases is provided, the method comprising: (i)stimulating peripheral blood cells, wherein the method comprisesstimulating peripheral blood T-cells with antigen presenting cells inthe presence of interleukin (IL)-7 and IL-15 and optionally in theabsence of IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV; (ii) stimulating T-cells obtained from (i)with antigen presenting cells in the presence of interleukin (IL)-7 andIL-15 and optionally in the absence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV, wherein (ii)is optionally repeated one or more times; and (iii) stimulating T-cellsobtained from (ii) with antigen presenting cells in the presence ofinterleukin (IL)-7 and IL-15, and in the presence of co-stimulatorycells, wherein the antigen presenting cells were previously exposed toone or more peptides, wherein the peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV, wherein (iii) is optionally repeated one or more times.

In any method of the disclosure, the HPV may be HPV16, HPV18, HPV1, HPV2and HPV3. Peptides comprising sequence that corresponds to one or moreof E1, E2, E3, E4, E5, E6, E7, L1, and L2 may be utilized in any methodof the disclosure. The peptides may comprise sequence that correspondsto: a) the HPV18 E6 protein and/or the HPV18 E7 protein, and/or b) theHPV16 E6 protein and/or the HPV16 E7 protein. In some cases, anindividual being provided with an effective amount of cells as describedherein has an HPV-associated disease, such as cancer, and the peptidescomprise sequence that corresponds to one or both of E6 and E7. Inspecific aspects, the one or more peptides comprises peptides of atleast or no more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 amino acids in length, and in particular the one or more peptidescomprises peptides of 15 amino acids in length. In specific embodiments,one or more peptides form a library and peptides in the library overlapin sequence with other peptides by 11 amino acids.

In particular embodiments, a therapeutically effective amount of T-cellsproduced by the method are provided to an individual that has beenexposed to HPV or that has HPV-associated disease. In specificembodiments, an HPV-associated disease comprises a neoplasm.

A therapeutically effective amount of T-cells produced by a method ofthe disclosure may be provided to an individual that has been exposed toHPV16, HPV18, or both, or that has HPV16-associated and/orHPV18-associated disease, including a neoplasm such as cancer.

In particular embodiments, the cancer is a cervical cancer, anal cancer,vulvar cancer, vaginal cancer, penile cancer, oropharyngeal cancer,nasopharyngeal carcinoma, laryngeal papillomatosis, laryngeal cancer,head and neck cancer, or a dysplasia of any site thereof.

In some cases, an individual that has received and/or will receive cellsof the disclosure has also received, is receiving, or will receive anadditional cancer therapy, such as surgery, radiation, hormone therapy,chemotherapy, immunotherapy, or a combination thereof.

In certain aspects, an individual that has received and/or will receivecells of the disclosure is determined as having HPV-associated cancer,such as HPV16-associated cancer or HPV18-associated cancer.

The following numbered paragraphs contain statements of broadcombinations of the inventive technical features herein disclosed:

-   -   1. A method for stimulating peripheral blood cells, the method        comprising stimulating peripheral blood T-cells with antigen        presenting cells in the presence of interleukin (IL)-7 and IL-15        and in the absence of IL-6 and/or IL-12, wherein the antigen        presenting cells were previously exposed to one or more        peptides, wherein the peptides comprise sequence that        corresponds to at least part of the sequence of one or more        proteins of human papillomavirus (HPV).    -   2. A method of producing therapeutic T-cells for HPV-associated        diseases, the method comprising the step of:        -   stimulating peripheral blood T-cells with antigen presenting            cells in the presence of one or more of IL-7 and IL-15 and            in the absence of IL-6 and/or IL-12, wherein the antigen            presenting cells were previously exposed to one or more            peptides, wherein the peptides comprise sequence that            corresponds to at least part of the sequence of one or more            proteins of HPV,        -   wherein the stimulating produces T-cells therapeutic for            HPV-associated diseases.    -   3. The method of paragraph 1 or 2, wherein the peripheral blood        T-cells are obtained from a prior stimulation of peripheral        blood cells.    -   4. The method of paragraph 3, wherein the prior stimulation of        peripheral blood cells comprises stimulating peripheral blood        cells with antigen presenting cells in the presence of IL-7 and        IL-15, and in the presence of IL-6 and/or IL-12, wherein the        antigen presenting cells were previously exposed to one or more        peptides, wherein the peptides comprise sequence that        corresponds to at least part of the sequence of one or more        proteins of HPV.    -   5. The method of paragraph 1 or 2, wherein prior to stimulating        said peripheral blood T-cells, the method further comprises        stimulating peripheral blood cells with antigen presenting cells        in the presence of IL-7 and IL-15, and in the presence of IL-6        and/or IL-12, wherein the antigen presenting cells were        previously exposed to one or more peptides, wherein the peptides        comprise sequence that corresponds to at least part of the        sequence of one or more proteins of HPV, to produce peripheral        blood T-cells.    -   6. The method of any one of paragraphs 1-7, wherein the antigen        presenting cells are dendritic cells, B-blasts, or PBMCs.    -   7. The method of any one of paragraphs 1-6, wherein the        peripheral blood T-cells are present in a population of        peripheral blood mononuclear cells (PBMCs) or are obtained or        isolated therefrom.    -   8. The method of paragraph 7, wherein the PBMCs in the        population are non-adherent PBMCs.    -   9. A method for stimulating T-cells specific for HPV or for an        HPV antigen, the method comprising stimulating T-cells specific        for HPV or for an HPV antigen with antigen presenting cells in        the presence of IL-7 and IL-15 and in the presence of        co-stimulatory cells, wherein the antigen presenting cells were        previously exposed to one or more peptides, wherein the peptides        comprise sequence that corresponds to at least part of the        sequence of one or more proteins of HPV.    -   10. A method of producing therapeutic T-cells for HPV-associated        diseases, the method comprising the step of stimulating T-cells        specific for HPV or for an HPV antigen with antigen presenting        cells in the presence of one or more of IL-7 and IL-15 and in        the presence of co-stimulatory cells, wherein the antigen        presenting cells were previously exposed to one or more        peptides, wherein the peptides comprise sequence that        corresponds to at least part of the sequence of one or more        proteins of HPV, wherein the stimulating produces T-cells        therapeutic for HPV-associated diseases.    -   11. The method of paragraph 9 or 10, wherein the antigen        presenting cells are activated T cells, dendritic cells,        B-blasts, or PBMCs.    -   12. The method of any one of paragraphs 9 to 11, wherein the        co-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+        cells, OX40+ cells, or a combination thereof.    -   13. A method of producing therapeutic T-cells for HPV-associated        diseases, the method comprising:        -   (i) stimulating peripheral blood cells, wherein the method            comprises stimulating peripheral blood T-cells with antigen            presenting cells in the presence of IL-7 and IL-15 and            optionally in the absence of IL-6 and/or IL-12, wherein the            antigen presenting cells were previously exposed to one or            more peptides, wherein the peptides comprise sequence that            corresponds to at least part of the sequence of one or more            proteins of HPV; and        -   (ii) stimulating T-cells obtained from (i) with antigen            presenting cells in the presence of IL-7 and IL-15, and in            the presence of co-stimulatory cells, wherein the antigen            presenting cells were previously exposed to one or more            peptides, wherein the peptides comprise sequence that            corresponds to at least part of the sequence of one or more            proteins of HPV.    -   14. The method of paragraph 13, wherein prior to step (ii)        T-cells obtained from (i) may be re-stimulated in the presence        of IL-7 and IL-15 but not in the presence of co-stimulatory        cells.    -   15. The method of paragraph 13 or 14, wherein the antigen        presenting cells used in (i) are dendritic cells (DC), B-Blasts        (BB) or PBMCs.    -   16. The method of any one of paragraphs 13 to 15, wherein the        antigen presenting cells used in (ii) are activated T cells,        dendritic cells (DC) or B-Blasts (BB).    -   17. The method of any one of paragraphs 13 to 16, wherein the        co-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+        cells, OX40+ cells, or a combination thereof.    -   18. A method of producing therapeutic T-cells for HPV-associated        diseases is provided, the method comprising:        -   (i) stimulating peripheral blood cells, wherein the method            comprises stimulating peripheral blood T-cells with antigen            presenting cells in the presence of interleukin (IL)-7 and            IL-15 and optionally in the absence of IL-6 and/or IL-12,            wherein the antigen presenting cells were previously exposed            to one or more peptides, wherein the peptides comprise            sequence that corresponds to at least part of the sequence            of one or more proteins of HPV;        -   (ii) stimulating T-cells obtained from (i) with antigen            presenting cells in the presence of interleukin (IL)-7 and            IL-15 and optionally in the absence of IL-6 and/or IL-12,            wherein the antigen presenting cells were previously exposed            to one or more peptides, wherein the peptides comprise            sequence that corresponds to at least part of the sequence            of one or more proteins of HPV, wherein (ii) is optionally            repeated one or more times; and        -   (iii) stimulating T-cells obtained from (ii) with antigen            presenting cells in the presence of interleukin (IL)-7 and            IL-15, and in the presence of co-stimulatory cells, wherein            the antigen presenting cells were previously exposed to one            or more peptides, wherein the peptides comprise sequence            that corresponds to at least part of the sequence of one or            more proteins of HPV, wherein (iii) is optionally repeated            one or more times.    -   19. The method of paragraph 18, wherein the antigen presenting        cells used in (i) and (ii) are DC, BB, or PBMCs.    -   20. The method of paragraph 18 or 19, wherein the antigen        presenting cells used in (iii) are activated T cells, DC, BB, or        PBMCs.    -   21. The method of any one of paragraphs 18 to 20, wherein the        co-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+        cells, OX40+ cells or a combination thereof.    -   22. The method of any one of paragraphs 1-21, wherein the HPV is        HPV16 or HPV18.    -   23. The method of any one of paragraphs 1-22, wherein the        peptides comprise sequence that corresponds to one or more of        E1, E2, E3, E4, E5, E6, E7, L1, and L2.    -   24. The method of any one of paragraphs 1-23, wherein the        HPV-associated disease is cancer and the peptides comprise        sequence that corresponds to one or both of E6 and E7.    -   25. The method of any one of paragraphs 1-25, wherein the        peptides comprise sequence that corresponds to:        -   a) the HPV18 E6 protein and/or the HPV18 E7 protein, and/or        -   b) the HPV16 E6 protein and/or the HPV16 E7 protein.    -   26. The method of any one of paragraphs 1-25, wherein the one or        more peptides comprises peptides of at least or no more than 8,        9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in        length.    -   27. The method of any one of paragraphs 1-26, wherein the one or        more peptides comprises peptides of 15 amino acids in length.    -   28. The method of any one of paragraphs 1-27, wherein one or        more peptides form a library and peptides in the library overlap        in sequence with other peptides by 11 amino acids.    -   29. The method of any one of paragraphs 1-28, wherein a        therapeutically effective amount of T-cells produced by the        method are provided to an individual that has been exposed to        HPV or that has HPV-associated disease.    -   30. The method of paragraph 29, wherein the HPV-associated        disease comprises a neoplasm.    -   31. The method of any one of paragraphs 1 to 30, wherein a        therapeutically effective amount of T-cells produced by the        method are provided to an individual that has been exposed to        HPV16, HPV18 or both, or that has HPV16-associated and/or        HPV18-associated disease.    -   32. The method of paragraph 31, wherein the HPV16-associated        and/or HPV18-associated disease comprises a neoplasm.    -   33. The method of paragraph 31 or 32, wherein the neoplasm is        cancer.    -   34. The method of paragraph 33, wherein the cancer is cervical        cancer, anal cancer, vulvar cancer, vaginal cancer, penile        cancer, oropharyngeal cancer, nasopharyngeal carcinoma,        laryngeal papillomatosis, laryngeal cancer, head and neck        cancer, or a dysplasia of any of site thereof.    -   35. The method of paragraph 33 or 34, wherein the individual has        received, is receiving, or will receive an additional cancer        therapy.    -   36. The method of paragraph 35, wherein the additional cancer        therapy is surgery, radiation, hormone therapy, chemotherapy,        immunotherapy, or a combination thereof.    -   37. The method of any one of paragraphs 33 to 36, wherein the        individual is determined as having HPV-associated cancer.    -   38. The method of any one of paragraphs 33 to 37, wherein the        individual is determined as having HPV16-associated cancer.    -   39. The method of any one of paragraphs 33 to 38, wherein the        individual is determined as having HPV18-associated cancer.    -   40. T-cells for use in a method of adoptive cellular        immunotherapy, wherein the T-cells are obtained by, obtainable        by, or are the product of, a method for stimulating peripheral        blood or T-cells or a method of producing therapeutic T-cells        according to any one of paragraphs 1 to 39, wherein the method        of adoptive cellular immunotherapy comprises administering the        T-cells to the subject.    -   41. Use of T-cells in the manufacture of a medicament for use in        a method of adoptive cellular immunotherapy comprising        administering the T-cells to the subject, wherein the T-cells        are obtained by, obtainable by, or are the product of, a method        for stimulating peripheral blood or T-cells or a method of        producing therapeutic T-cells according to any one of claims 1        to 39.    -   42. A method of preparing a pharmaceutical composition,        medicament or vaccine, the method comprising stimulating        peripheral blood or T-cells or producing therapeutic T-cells        according to any one of claims 1 to 39, and mixing the cells        obtained with a pharmaceutically acceptable carrier, adjuvant,        diluent or excipient.    -   43. A method of treating a cancer in a subject, the method        comprising:        -   (1) isolating T cells from a subject;        -   (2) generating or expanding a population of T cells specific            for a human papillomavirus (HPV) by a method comprising:            stimulating the T-cells with antigen presenting cells in the            presence of interleukin (IL)-7 and IL-15 and in the absence            of IL-6 and/or IL-12, wherein the antigen presenting cells            were previously exposed to one or more peptides, wherein the            peptides comprise sequence that corresponds to at least part            of the sequence of one or more proteins of HPV; and        -   (3) administering the generated or expanded population of T            cells to a subject.    -   44. The method of paragraph 43, wherein the T-cells stimulated        in (2) are obtained from a prior stimulation of peripheral blood        cells or T-cells.    -   45. The method of paragraph 43, wherein prior to stimulating        said T-cells, the method comprises stimulating peripheral blood        cells or T-cells with antigen presenting cells in the presence        of IL-7 and IL-15, and in the presence of IL-6 and/or IL-12,        wherein the antigen presenting cells were previously exposed to        one or more peptides, wherein the peptides comprise sequence        that corresponds to at least part of the sequence of one or more        proteins of HPV.    -   46. The method of paragraph 43, wherein after (2) and before (3)        the method comprises stimulating the T-cells obtained from (2)        with antigen presenting cells in the presence of IL-7 and IL-15,        and in the presence of co-stimulatory cells, wherein the antigen        presenting cells were previously exposed to one or more        peptides, wherein the peptides comprise sequence that        corresponds to at least part of the sequence of one or more        proteins of HPV.    -   47. The method of paragraph 46 wherein the co-stimulatory cells        are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells, or a        combination thereof.    -   48. The method of paragraph 43, wherein after (2) and before (3)        the method comprises (i) re-stimulating the T-cells obtained        from (2) in the presence of IL-7 and IL-15 but not in the        presence of co-stimulatory cells, and (ii) stimulating the        T-cells obtained after (i) with antigen presenting cells in the        presence of IL-7 and IL-15, and in the presence of        co-stimulatory cells, wherein the antigen presenting cells were        previously exposed to one or more peptides, wherein the peptides        comprise sequence that corresponds to at least part of the        sequence of one or more proteins of HPV.    -   49. The method of paragraph 43, wherein the antigen presenting        cells are dendritic cells (DC), B-blasts (BB), or peripheral        blood mononuclear cells (PBMCs).    -   50. The method of paragraph 43, wherein in (1) the T-cells are        isolated from a population of peripheral blood mononuclear cells        (PBMCs).    -   51. The method of paragraph 43, wherein the cancer is cervical        cancer, anal cancer, vulvar cancer, vaginal cancer, penile        cancer, oropharyngeal cancer, nasopharyngeal carcinoma,        laryngeal papillomatosis, laryngeal cancer, head and neck        cancer, or a dysplasia of any of site thereof.    -   52. The method of paragraph 43, wherein the cancer is        HPV-positive.    -   53. The method of paragraph 43, wherein the subject is        determined as having HPV-associated cancer.    -   54. A method of treating a cancer in a subject, the method        comprising:        -   (1) isolating T cells from a subject;        -   (2) generating or expanding a population of T cells specific            for a human papillomavirus (HPV) by a method comprising:        -   (i) stimulating the T-cells with antigen presenting cells in            the presence of interleukin (IL)-7 and IL-15, wherein the            antigen presenting cells were previously exposed to one or            more peptides, wherein the peptides comprise sequence that            corresponds to at least part of the sequence of one or more            proteins of HPV;        -   (ii) stimulating T-cells obtained from (i) with antigen            presenting cells in the presence of interleukin (IL)-7 and            IL-15 and in the absence of IL-6 and/or IL-12, wherein the            antigen presenting cells were previously exposed to one or            more peptides, wherein the peptides comprise sequence that            corresponds to at least part of the sequence of one or more            proteins of HPV, wherein (ii) is optionally repeated one or            more times; and        -   (iii) stimulating T-cells obtained from (ii) with antigen            presenting cells in the presence of interleukin (IL)-7 and            IL-15, and in the presence of co-stimulatory cells, wherein            the antigen presenting cells were previously exposed to one            or more peptides, wherein the peptides comprise sequence            that corresponds to at least part of the sequence of one or            more proteins of HPV, wherein (iii) is optionally repeated            one or more times.        -   (3) administering the generated or expanded population of T            cells to a subject.    -   55. The method of paragraph 54, wherein stimulation of T-cells        in (i) is in the presence of IL-6 and/or IL-12.    -   56. The method of paragraph 54, wherein the antigen presenting        cells used in (i) and (ii) are dendritic cells (DC), B-blasts        (BB), or peripheral blood mononuclear cells (PBMCs).    -   57. The method of paragraph 54, wherein the antigen presenting        cells used in (iii) are activated T cells, dendritic cells (DC),        B-blasts (BB), or peripheral blood mononuclear cells (PBMCs).    -   58. The method of paragraph 54, wherein the co-stimulatory cells        are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells or a        combination thereof.    -   59. A method of treating a cancer in a subject, the method        comprising:        -   (1) isolating T cells from a subject;        -   (2) generating or expanding a population of T cells specific            for a human papillomavirus (HPV) by a method comprising:            stimulating T-cells specific for HPV or for an HPV antigen            with antigen presenting cells in the presence of IL-7 and            IL-15 and in the presence of co-stimulatory cells, wherein            the antigen presenting cells were previously exposed to one            or more peptides, wherein the peptides comprise sequence            that corresponds to at least part of the sequence of one or            more proteins of HPV; and        -   (3) administering the generated or expanded population of T            cells to a subject.    -   60. The method of paragraph 59, wherein the antigen presenting        cells are activated T cells, dendritic cells (DC), B-blasts        (BB), or peripheral blood mononuclear cells (PBMCs).    -   61. The method of paragraph 59, wherein the co-stimulatory cells        are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells, or a        combination thereof.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A demonstrates a method in the art that utilizes certainconditions for the production of HPV16-specific T-cells. FIG. 1A is abar chart showing production of spot forming colonies (SFC) onstimulation of PBMCs from three HPV-associated cancer patients(identified as OCV, HND and HNC) with autologous DCs loaded with nopepmix (Neg), HPV16 E6 pepmix (HPV16 E6) or HPV16 E7 pepmix (HPV16 E7).For patient OCV the three bars present from left to right are Neg, HPV16E6 and HPV16 E7. For patients HND and HNC the two bars present from leftto right are HPV16 E6 and HPV16 E7.

FIG. 1B demonstrates a method of the disclosure that utilizes, undercertain novel conditions, the production of a mixture of T-cellsspecific for HPV16 or HPV18 by stimulation of T-cells in the presence ofIL-7 and IL-15 and in the absence of IL-6 and IL-12. FIG. 1B is a barchart showing production of spot forming colonies (SFC) on stimulationof PBMCs from three HPV-associated cancer patients (identified as OCV,PCV and HND). For patient OCV the five bars present from left to rightare no pepmix (Neg), HPV16 E6 pepmix (HPV16 E6), HPV16 E7 pepmix (HPV16E7), HPV18 E6 pepmix (HPV18 E6), and HPV18 E7 pepmix (HPV18 E7). Forpatient PCD the two bars present from left to right are HPV16 E6 andHPV16 E7. For patient HND the four bars present from left to right areHPV16 E6, HPV16 E7, HPV18 E6 and HPV18 E7.

FIG. 2 is a chart showing in vivo expansion and persistence of infusedHPV stimulated T-cells transduced with a dominant negative receptor forTGF-beta (DNRII) in patient #1 at time points post infusion.

FIG. 3 is a chart showing in vivo expansion and persistence of infusedHPV stimulated T cells transduced with a dominant negative receptor forTGF-beta (DNRII) in patient #2 at time points post infusion.

FIG. 4 shows PET scans (left and center) and photographs of physicalexamination (right) for patient #2. Top row: pre-treatment. Bottom row:6 weeks after treatment with HPV stimulated T cells produced accordingto the present invention.

FIGS. 5A and 5B are scatterplots showing surface expression of (FIG. 5A)CD83 and CD80, and (FIG. 5B) CCR7 and PD-L1 expression bymonocyte-derived DCs derived from Donor 1 CD14+ PBMCs following cultureaccording to experimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11(see Table 4).

FIGS. 6A and 6B are scatterplots showing surface expression of CCR7 andCD4SRO by (FIG. 6A) CD4+ T cells, and (FIG. 6B) CD8+ T cells obtainedfollowing stimulation of autologous CD14− PBMCs derived from Donor 1 for9 days with EBV peptide-pulsed mature DCs cultured according toexperimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 (see Table4).

FIG. 7 is a bar chart showing the total number of virus-specific T cellsobtained following stimulation of autologous CD14- PBMCs derived fromDonor 1 for 9 days with EBV peptide-pulsed mature DCs cultured accordingto experimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 (seeTable 4).

FIG. 8 is a bar chart shows the proportions of IFNγ+CD8+ CTLs andIFNγ+CD4+ Th cells (background subtracted) obtained followingstimulation of autologous CD14− PBMCs derived from Donor 1 for 9 dayswith EBV peptide-pulsed mature DCs cultured according to experimentalconditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 (see Table 4).

FIG. 9 is a bar chart showing the proportions of EBV antigen-reactiveIFNγ+CD8+ CTLs and IFNγ+CD4+ Th cells (background subtracted) obtainedfollowing stimulation of autologous CD14− PBMCs derived from Donor 1 for9 days with EBV peptide-pulsed mature DCs cultured according toexperimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 (see Table4).

FIG. 10 is a graph showing the total number of virus-specific T cellsplotted against the proportion of IFNγ+CD8+ CTLs obtained followingstimulation of autologous CD14− PBMCs derived from Donor 1 for 9 dayswith EBV peptide-pulsed mature DCs cultured under different experimentalconditions (see Table 4).

FIGS. 11A and 11B are scatterplots showing surface expression of (FIG.11A) CD83 and CD80, and (FIG. 11B) CCR7 and PD-L1 expression bymonocyte-derived DCs derived from Donor 2 CD14+ PBMCs following cultureaccording to experimental conditions 1, 2, 3, 12, 13, 14, 15, 16, 17,18, 19 or 20 (see Table 4).

FIGS. 12A and 12B are scatterplots showing surface expression of CCR7and CD45RO by (FIG. 12A) CD4+ T cells, and (FIG. 12B) CD8+ T cellsobtained following stimulation of autologous CD14− PBMCs derived fromDonor 2 for 9 days with EBV peptide-pulsed mature DCs cultured accordingto experimental conditions 1, 2, 3, 12, 13, 14, 15, 16, 17, 18, 19 or 20(see Table 4).

FIG. 13 is a bar chart showing the total number of virus-specific Tcells obtained following stimulation of autologous CD14− PBMCs derivedfrom Donor 2 for 9 days with EBV peptide-pulsed mature DCs culturedaccording to experimental conditions 1, 2, 3, 12, 13, 14, 15, 16, 17,18, 19 or 20 (see Table 4).

FIG. 14 is a bar chart shows the proportions of IFNγ+CD8+ CTLs andIFNγ+CD4+ Th cells (background subtracted) obtained followingstimulation of autologous CD14− PBMCs derived from Donor 2 for 9 dayswith EBV peptide-pulsed mature DCs cultured according to experimentalconditions 1, 2, 3, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (see Table 4).

FIG. 15 is a bar chart showing the proportions of EBV antigen-reactiveIFNγ+CD8+ CTLs and IFNγ+CD4+ Th cells (background subtracted) obtainedfollowing stimulation of autologous CD14− PBMCs derived from Donor 2 for9 days with EBV peptide-pulsed mature DCs cultured according toexperimental conditions 1, 2, 3, 12, 13, 14, 15, 16, 17, 18, 19 or 20(see Table 4).

FIG. 16 is a graph showing the total number of virus-specific T cellsplotted against the proportion of IFNγ+CD8+ CTLs obtained followingstimulation of autologous CD14-PBMCs derived from Donor 2 for 9 dayswith EBV peptide-pulsed mature DCs cultured under different experimentalconditions (see Table 4).

FIGS. 17A, 17B, 17C, 17D and 17E are scatterplots and bar charts showingsurface expression of (FIGS. 17A) CD80 and CD83, and (FIG. 17B) CCR7 andPD-L1 by monocyte-derived DCs derived from three different donorsfollowing culture according to the experimental conditions 1, 2, 5, 18,19 and 20 (see Table 4). FIG. 17C shows the proportions of CD80+CD83−cells, FIG. 17D shows the proportions of CD80+CD83+ cells, and FIG. 17Eshows the proportions of PD-L1+ cells.

FIG. 18 is a bar chart showing the total number of virus-specific Tcells obtained following stimulation of autologous CD14− PBMCs derivedfrom the three different donors for 9 days with EBV peptide-pulsedmature DCs cultured according to the experimental conditions 2, 5, 18,19 and 20 (see Table 4).

FIG. 19 is a bar chart showing the proportions of IFNγ+CD8+ CTLs andIFNγ+CD4+ Th cells (background subtracted) obtained followingstimulation of autologous CD14− PBMCs derived from the different donorsfor 9 days with EBV peptide-pulsed mature DCs cultured according to theexperimental conditions 2, 5, 18, 19 and 20 (see Table 4).

FIG. 20 shows the proportions of EBV antigen-reactive IFNγ+CD8+ CTLs andIFNγ+CD4+ Th cells (background subtracted) obtained followingstimulation of autologous CD14− PBMCs derived from the different donorsfor 9 days with EBV peptide-pulsed mature DCs cultured according to theexperimental conditions 2, 5, 18, 19 and 20 (see Table 4).

FIG. 21 is a graph showing the scaled total number of virus-specific Tcells and the scaled frequency of IFNγ+ T cells obtained followingstimulation of autologous CD14-PBMCs derived from the three donors for 9days with EBV peptide-pulsed mature DCs cultured under differentexperimental conditions (see Table 4).

FIGS. 22A and 22B are graphs showing the results of two separateexperiments investigating overall fold expansion of cells followingstimulations using K562-cs cells as costimulatory cells as compared toULCL clones #5 and #13.

FIGS. 23A and 23B are graphs showing the results of two separateexperiments investigating overall fold expansion of virus-specific Tcells following stimulations using K562-cs cells as costimulatory cellsas compared to ULCL clones #5 and #13, as determined by ELISPOT analysisof the number of IFNγ producing cells.

FIGS. 24A, 24B and 24C provide scatterplots from a representative donor(n=7) showing the proportions of (FIG. 24A) CD3-CD56+ NK cells, (FIG.24B) CD4+ T cells and CD8+ T cells, and (FIG. 24C) gamma delta T cellsand alpha beta T cells in populations expanded using K562-cs cells orULCL clones #5 and #13 as costimulatory cells.

FIG. 25 provides histograms showing representative results ofcharacterization of gamma delta TCR expression by ULCL clones #5 and#13, as compared to expression by K562cs cells.

FIGS. 26A and 26B are bar charts for two different donors showing thefrequency of virus-specific T cells in populations expanded usingK562-cs cells or ULCL clones #5 and #13 as costimulatory cells, afterthe indicated number of stimulations, as determined by ELISPOT analysisof the number of IFNγ producing cells.

FIGS. 27A, 27B, 27C and 27D are bar charts for four different donorsshowing the frequency of T cells specific for the indicated EBVantigens, after the indicated number of stimulations using K562cs cells(K), ULCL clone #5 (5) or LCLs (L) as costimulatory cells, as determinedby ELISPOT analysis of the number of IFNγ producing cells.

FIGS. 28A, 28B, 28C and 28D are bar charts and graphs showing thefrequency and fold expansion of virus-specific T cells in populationsexpanded using K562-cs cells or ULCL clones #4, #5 and #13 ascostimulatory cells, after the indicated number of stimulations, asdetermined by ELISPOT analysis of the number of IFNγ producing cells.

FIGS. 29A and 29B are bar charts showing the frequency of virus-specificT cells in populations expanded using ULCL clone #4 or parental ULCLcells as costimulatory cells, after the indicated number ofstimulations, as determined by ELISPOT analysis of the number of IFNγproducing cells.

FIGS. 30A and 30B are bar charts for two different donors showing thefrequency of virus-specific T cells in populations expanded using ULCLclone #5 or K562cs cells as costimulatory cells, after the indicatednumber of stimulations, as determined by ELISPOT analysis of the numberof IFNγ producing cells.

FIGS. 31A and 31B are bar charts for two different donors showing thefold expansion of virus-specific T cells in populations expanded usingULCL clone #5 or K562cs cells as costimulatory cells, after theindicated number of stimulations, as determined by ELISPOT analysis ofthe number of IFNγ producing cells.

FIG. 32 provides scatterplots showing surface expression of CD3 and CD56by cells expanded using ULCL clone #5 or K562cs cells as costimulatorycells, after the indicated number of stimulations, as determined by flowcytometry, for donor PB.

FIG. 33 provides scatterplots showing surface expression of CD3 and CD56by cells expanded using ULCL clone #5 or K562cs cells as costimulatorycells, after the indicated number of stimulations, as determined by flowcytometry, for donor KP.

FIG. 34 provides scatterplots showing surface expression of CD45RO andCCR7 by cells expanded using ULCL clone #5 or K562cs cells ascostimulatory cells, after the indicated number of stimulations, asdetermined by flow cytometry, for donor PB.

FIG. 35 provides scatterplots showing surface expression of CD45RO andCCR7 by cells expanded using ULCL clone #5 or K562cs cells ascostimulatory cells, after the indicated number of stimulations, asdetermined by flow cytometry, for donor KP.

FIGS. 36A and 36B are bar charts for two different donors showing thefrequency of virus-specific T cells in populations expanded using ULCLclone #5 or K562cs cells as costimulatory cells, after the indicatednumber of stimulations, as determined by ELISPOT analysis of the numberof IFNγ producing cells.

FIGS. 37A and 37B are bar charts for two different donors showing thefold expansion of virus-specific T cells in populations expanded usingULCL clone #5 or K562cs cells as costimulatory cells, after theindicated number of stimulations, as determined by ELISPOT analysis ofthe number of IFNγ producing cells.

FIG. 38 provides scatterplots showing surface expression of CD3 and CD56by cells expanded using ULCL clone #5 or K562cs cells as costimulatorycells, after the indicated number of stimulations, as determined by flowcytometry, for one donor.

FIG. 39 provides scatterplots showing surface expression of CD3 and CD56by cells expanded using ULCL clone #5 or K562cs cells as costimulatorycells, after the indicated number of stimulations, as determined by flowcytometry, for one donor.

FIG. 40 provides scatterplots showing surface expression of CD45RO andCCR7 by cells expanded using ULCL clone #5 or K562cs cells ascostimulatory cells, after the indicated number of stimulations, asdetermined by flow cytometry, for one donor.

FIG. 41 provides scatterplots showing surface expression of CD45RO andCCR7 by cells expanded using ULCL clone #5 or K562cs cells ascostimulatory cells, after the indicated number of stimulations, asdetermined by flow cytometry, for one donor.

FIG. 42 is a bar chart showing the frequency of HPV-specific T cells inpopulations expanded using ULCL clone #5 or K562cs cells ascostimulatory cells in stimulations comprising the indicated cytokines,and after the indicated number of stimulations, as determined by ELISPOTanalysis of the number of IFNγ producing cells.

FIG. 43 is a graph showing the fold expansion of cells in populationsexpanded using ULCL clone #5 or K562cs cells as costimulatory cells instimulations comprising the indicated cytokines, and after the indicatednumber of stimulations.

FIG. 44 provides scatterplots showing surface exspression of CD3 andCD56 by cells expanded using ULCL clone #5 or K562cs cells ascostimulatory cells in stimulations comprising the indicated cytokines,and after the indicated number of stimulations, as determined by flowcytometry.

FIG. 45 provides scatterplots showing surface expression of CD4 and CD8by cells expanded using ULCL clone #5 or K562cs cells as costimulatorycells in stimulations comprising the indicated cytokines, and after theindicated number of stimulations, as determined by flow cytometry.

FIG. 46 provides scatterplots showing surface expression of CD45RO andCCR7 by cells expanded using ULCL clone #5 or K562cs cells ascostimulatory cells in stimulations comprising the indicated cytokines,and after the indicated number of stimulations, as determined by flowcytometry.

FIG. 47 is a schematic illustration of general embodiments ofvirus-specific T-cell (VST) generation methods of the disclosure.

FIG. 48 provides bar charts demonstrating improved specificity ofmethods of the disclosure that employ IL-7 and IL-15 as compared toknown methods that employ IL-4 and IL-7.

FIG. 49 is a bar chart showing improved EBV antigen specificity oflymphoma patient EBVSTs obtained by stimulations performed in thepresence of IL-7 and IL-15, as determined by ELISPOT.

FIG. 50 is a bar chart demonstrating that stimulation in the presence ofhigh doses of IL-15 increases frequency of VSTs.

FIG. 51 is a bar chart showing shows that stimulation in the presence ofhigh doses of IL-15 increases the proportion of central memory EBVSTs.

FIG. 52 provides bar charts showing excessive NK-cell outgrowth inEBVSTs from some patients.

FIG. 53 is a schematic illustration of the generation ofpepmix-activated EBVSTs from CD45RA+ cell-depleted PBMCs.

FIG. 54 is a bar chart showing that CD45RA depletion decreases thefrequency of CD3-CD56+ NK cells in EBVSTs expanded from healthy donors.

FIG. 55 is a bar chart showing that removal of CD45RA+ cells increasesproliferation of EBVSTs.

FIG. 56 is a graph that demonstrates that CD45RA depletion enhances thefold expansion of EBVSTs.

FIG. 57 is a bar chart showing that CD45RA depletion enhances antigenspecificity of EBVSTs at the end of a second stimulation (at day 16).

FIG. 58 is a bar chart which demonstrates that CD45RA depletion enhancesantigen specificity of EBVSTs.

FIG. 59 is a bar chart which demonstrates increased antigen specificityof CD45RA depleted EBVSTs is sustained after a third stimulation.

FIG. 60 is a bar chart which demonstrates that CD45RA depletiondecreases NK cell population outgrowth in lymphoma patient EBVSTs.

FIG. 61 is a bar chart showing that CD45RA depletion increases thefrequency of antigen specific T-cells in lymphoma patient EBVSTs.

FIG. 62 is a bar chart demonstrating that CD45RA depletion increasesantigen specificity in EBVSTs from lymphoma patients.

FIG. 63 is a bar chart showing the effect of CD45RA depletion onproliferation of lymphoma patients' EBVSTs.

FIG. 64 is a chart which demonstrates that CD45RA-depletion enhancescytolytic activity against pepmix-pulsed autologous activated T-cells(aATCs).

DETAILED DESCRIPTION

The scope of the present application is not intended to be limited tothe particular embodiments of the process, machine, manufacture,composition of matter, means, methods and steps described in thespecification.

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The present disclosure concerns the production and use of therapeuticT-cells for individuals that are in need of HPV-specific T-cells, e.g.,HPV16- and/or HPV18-specific T-cells, including for treating HPVinfection and HPV-associated medical conditions. In particularembodiments, the methods and compositions are useful for treatingneoplasms that are indirectly or directly related to HPV infection, andsuch neoplasms may be benign or malignant. Between 13 and 18 HPV strainshave been characterized as conferring a high oncogenic risk, with 12 ofthese strains belonging to the HPV species 7 (HPV-18, -39, -45, -59,-68) and species 9 (HPV-16, -31, -33, -35, -52, -58, -67). HPV Types 6and 11 cause laryngeal papillomatisis.

I. HPV Antigen(s) and Generation of Pepmixes

Methods of the disclosure utilize antigen-presenting cells that presentmixtures of peptides to T-cells. Such “loaded” APCs are generated priorto exposure to peripheral blood T-cells for stimulation of theperipheral blood T-cells, and the generation of the loaded APCs may ormay not be performed by the individual or entity that performs thestimulation step for the peripheral blood T-cells. Thus, in someembodiments, an effective amount of a library of peptides is provided toAPCs as part of methods that ultimately generate therapeutic CTLs. Inmethods of the disclosure, prior to a stimulation step, APCs are exposedto a sufficient amount of the library of peptides. The library, inparticular cases, comprises a mixture of peptides (“pepmixes”) that spanpart or all of the same antigen, although in some cases the librarycomprises pepmixes that span part or all of one or more antigens, andthe one or more antigens may or may not be from the same HPV. Inparticular embodiments, peptides for the APCs are non-natural, and theymay or may not be chemically synthesized or produced by recombinantmeans.

In utilizing a library of mixtures of peptides from one or more HPVantigens, the various peptides may come from any part of a givenprotein, but in specific cases the peptides collectively span the lengthof the majority or all of the protein, wherein the sequence of thepeptides overlap at least in part to facilitate coverage of the entiredesired region of the specific antigen(s). In some cases the peptidesspan the length of one or more known epitopes or domains of therespective antigen to which the peptides correspond. Certain regions maybe covered by peptides that span the length of the region, including aregion such as a N-terminal domain, C-terminal domain, extracellulardomain, or intracellular domain, for example.

The antigens from which the peptides are derived may be antigens forHPVs that may be of any kind, but in specific embodiments the antigensare such that they allow for direction of cytotoxic T-cells toneoplasms, including cancers, associated with HPV infection. Inparticular embodiments, the peptides are derived from, or have sequencethat corresponds to, at least part of one or more antigens of at leastone HPV type, including HPV16 and/or HPV18. For example, in late stagecervical cancer, the HPV virus integrates into a tumor cell genome andloses all of its other genes except E6 and E7, so in some cases theseantigens are targeted. In embodiments wherein one would treat an earlierstage of cancer, such as before the virus integrated, one could utilizepeptides from antigens other than E6 and E7, including E5 and L1 and L2,for example. However, given that the two primary oncoproteins of highrisk HPV types are E6 and E7, in specific embodiments the sequence ofthe peptides are obtained from E6 and/or E7 from any HPV, but HPV16and/or HPV18, in particular. Peptides from any of antigens E1, E2, E3,E4, E5, E6, E7, L1, and/or L2 may be utilized in methods of thedisclosure.

In some cases, a pepmix library includes peptides corresponding to oneor more antigens from a single type of HPV virus, and those peptides mayor may not provide sequence coverage across the entire antigen(s) inquestion. In other cases, a pepmix library includes peptidescorresponding to one or more antigens from more than one HPV virus, andthose peptides may or may not provide sequence coverage across theentire antigen(s) in question. The pepmix may or may not be enriched forpeptides corresponding to one or more certain regions of one or morecertain antigens or corresponding to the entirety of one or more certainantigens.

Pepmixes utilized in the disclosure may be from commercially availablepeptide libraries or may be synthetically generated, for example.Examples of available libraries include those from JPT Technologies(Springfield, Va.) or Miltenyi Biotec (Auburn, Calif.). The skilledartisan, based on known sequences of HPV16 E6, HPV16 E7, HPV18 E6, andHPV18 E7, for example, would have sufficient information to be able togenerate peptides that correspond to their exemplary, respectivesequences. An example of sequence of the HPV16 E6 protein is availableat the National Center for Biotechnology Information's GenBank® databaseat GenBank® Accession No. AIQ82776.1 GI:688010703. An example ofsequence of the HPV16 E7 protein is at GenBank® Accession No. AIQ82814.1GI:688010789. An example of sequence of the HPV18 E6 protein is atGenBank® Accession No. AGU90423.1 GI:537801975. An example of sequenceof the HPV18 E7 protein is at GenBank® Accession No. AGU90424.1GI:537801976.

In particular embodiments, a library is comprised of peptides of acertain length that correspond to their respective antigens, although insome cases a library is comprised of a mixture of peptides with two ormore different lengths. The peptides may be of a certain length(s) andthey may overlap in sequence of a certain amount, although there may bevariability of length of overlap in some libraries. In particularembodiments, the peptides are at least 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,or 35 or more amino acids in length, for example. In particularembodiments, there is overlap among the peptides of at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids in length, forexample. In specific embodiments, the peptides are 15 amino acids longand overlap one another by 11 amino acids. A mixture of differentpeptides may include any ratio of the different peptides, although insome embodiments each particular peptide is present at substantially thesame numbers in the mixture as another particular peptide. Althoughcoverage of an antigen in sequence for the peptides may be random andsubstantially even over a given region of an antigen, in someembodiments a library may be enriched for one or more particularpeptides, such as one or more peptides that are known to encode anepitope or a part thereof, for example.

In particular embodiments, the pepmix for a particular antigen proteincomprise all possible HLA class I epitopes that are 8 to 10 amino acidslong, for example. In specific embodiments, longer peptides are utilizedto cover all class II epitopes for a particular peptide. In certainaspects, the peptides are at a maximum of 30 amino acids in length withoverlapping of 25 amino acids.

II. Methods of Producing and Using Therapeutic T-Cells

A. Producing Therapeutic T-Cells

In certain aspects, the present disclosure concerns the development ofimmune cells, such as cytotoxic T-cells, that target one or moreantigens from at least one HPV virus.

Methods disclosed herein may involve the stimulation and/or expansion ofimmune cells. The methods may involve the stimulation and/or expansionof peripheral blood cells, such as peripheral blood mononuclear cells.The methods may involve expansion of an immune cell population (e.g. apopulation of T-cells) from within a population of immune cells (e.g.PBMCs). For example, a population of T-cells may be expanded from withina population of PBMCs, by stimulation of the T-cells within thepopulation of PBMCs. Accordingly, in embodiments of the methodsdisclosed herein, stimulation and/or expansion of T cells may involvestimulation of a population of PBMCs. In some embodiments, a populationof T-cells may be expanded from within a population oftumor-infiltrating lymphocytes, by stimulation of the T-cells within thepopulation of tumor-infiltrating lymphocytes. Accordingly, inembodiments of the methods disclosed herein, stimulation and/orexpansion of T cells may involve stimulation of a population oftumor-infiltrating lymphocytes. In some embodiments, a population ofT-cells may be expanded from within a population of T-cells (e.g. apopulation of T cells of heterogeneous specificity), which may have beenobtained from a blood sample, a population of PBMCs, or from apopulation of tumor-infiltrating lymphocytes. Thestimulations/expansions may result in an increase the numberHPV-specific immune cells (e.g. HPV-specific T cells, such asHPV-specific CTLs), and/or result in an increased proportion of suchcells in the cell population at the end of the stimulation/expansion.The methods may involve the stimulation and/or expansion of T cells. Thecells may have been obtained from the patient to be treated (i.e.,autologous cells), or from another individual (i.e., allogeneic cells).The methods involve stimulation and/or expansion of isolated immunecells, in certain embodiments. That is, specific methods may beperformed on a population of cells that contains substantially nonon-immune cells, such as erythrocytes. In some cases, the immune cellsare isolated PBMCs, or isolated T cells. The cells may have beenobtained from a sample of blood, such as a sample of blood obtained fromthe patient or individual. The cells may have been obtained from tissuesample or biopsy. The cells may have been obtained from a tumor (e.g.tumor-infiltrating lymphocytes). Certain methods disclosed hereininvolve a step of obtaining PBMCs and/or T cells from a sample obtainedfrom the patient. Certain embodiments of methods do not involve the stepof obtaining a sample of blood or cells from the patient or individual,but instead are performed on a sample or cells that have been previouslyobtained. The method may involve processing the sample, such asenriching the sample for immune cells, such as PBMCs and/or T cells.Such methods may involve removing or substantially reducing the amountof, erythrocytes, platelets, serum and/or plasma in a sample. This mayresult in a population of immune cells containing substantially no othercells, such as erythrocytes. Methods disclosed herein may be performedon isolated immune cells, or a sample containing immune cells inaddition to other cells.

In methods of producing the T-cells, peripheral blood T-cells may beinitially stimulated with APCs that have been exposed to one or morepeptides that span some or all of at least one HPV antigen. Theantigenic peptides may be provided to the APCs in a library of peptidemixtures, and multiple libraries of pepmixes may be provided to the samecollection of APCs. In some embodiments, the collection includes bothimmunodominant and subdominant antigens.

In embodiments of the disclosure, therapeutic T-cells are generated andmay be provided to an individual that has an HPV infection or is at riskof having an HPV-associated medical condition that results indirectly ordirectly from an HPV infection. In methods of producing the therapeuticT-cells, under certain conditions peripheral blood T-cells are mixedwith APCs that are loaded with a library of peptides that span part orall of one or more antigens, including part or all of a HPV16 and/orHPV18 antigen, including E6 and/or E7, for example. In specificembodiments, for the stimulating step the T-cells reside within apopulation of PBMCs.

In some embodiments, the APCs used in certain steps may be dendriticcells (DCs). Methods for generation of DCs are well known in the art,e.g. see Ramos et al., supra. Monocytes may be isolated from PBMCs byCD14 selection and cultured in DC medium and 2 mM alanyl-glutamine with800 U/ml granulocyte/macrophage colony stimulating factor (GM-CSF) and1000 U/ml interleukin 4 (IL-4) for 5 days. GM-CSF and IL-4 may bereplenished on day 3. On day 5, DCs are matured in DC media with 10ng/ml interleukin-1β(IL-1β), 100 ng/ml interleukin 6 (IL-6), 10 ng/mlprostaglandin E2, 800 U/ml GM-CSF and 1000 U/ml IL-4. DC maturation maybe assessed by flow cytometry to detect upregulation of CD80. CD83, CD86and HLA-DR.

In some embodiments, the APCs used in certain steps are activatedT-cells. Activated T-cells may be polyclonal T-cells (T-APCs) generatedusing a portion of the autologous PBMC isolated from the venesectedblood. The cells may be activated by culturing in cell culture platesthat are coated with anti-CD3 and anti-CD28 antibodies. The cells arethen cultured to expand in the presence of IL-2 for 2 weeks. Theexpanded T-APC can be cryopreserved for later use. 2-3 days prior tousing T-APC for stimulation (e.g., for the 3rd cycle of stimulation andoptionally for subsequent stimulations), cryopreserved cells are thawedand re-stimulated in anti-CD3 and anti-CD28 antibody-coated cell cultureplates. On the day of stimulation, the T-APC cells are harvested andpulsed with the HPV E6/E7 peptides, followed by adding to the on-goingculture of HPV stimulated T-cells at 1:1 ratio.

In some embodiments, the APCs used in certain steps and/or methods maybe B-blasts (BBs). B-blasts may be generated from a patient's autologousPBMC, for example. The B lymphocytes within the PBMCs are activated byco-culturing with an irradiated allogeneic CD40L-expressing MRCSepithelial cell line and expanded in media containing 100 U/ml IL-4 and1 microgram/ml cyclosporin A.

In some embodiments, there is a method of generating T-cells that targetat least one antigen from one or both of HPV16 and HPV18, and thisoccurs generally by contacting a plurality of PBMCs with a plurality ofAPCs loaded for peptides from a library of peptides that correspond toone or more particular HPV16 and/or HPV18 viral antigens. In specificembodiments, the exposure of the two populations of cells allows forexpansion of the T-cells. In particular embodiments, the stimulationstep(s) occurs in the presence of one or more particular cytokines,which may be mammalian (e.g. murine, human) or human cytokines. Incertain embodiments, the one or more cytokines are IL-7 and IL-15,although in alternative embodiments the cytokine(s) are selected fromthe group consisting of IL-15, IL-7, IL-21, IL-12, IL-6, IL-4, and acombination thereof. In specific embodiments, one or more steps of themethods do not occur in the presence of IL-2, IL-4, IL-6, IL-7, IL-12,and/or IL-21, although alternatively IL-2, IL-4, IL-6, IL-7, IL-12,and/or IL-21 may be utilized. Reference to the presence of a cytokine isto presence of exogenously added cytokine, i.e. excluding any cytokinepresent within or secreted by the culture of cells. In some embodiments,the peptides are further defined as peptides that overlap in sequence tospan part or all of a HPV antigen. For example, in certain aspects thepeptides overlap by at least 10 amino acids, and particularly 11, and insome embodiments the peptides are at least 12 or more amino acids inlength, and particularly 15 amino acids in length.

The selection of an appropriate amount or concentration of a givencytokine for inclusion in a cell culture is within the ability of theperson or ordinary skill in the art. By way of example, the following isa list of certain interleukins and examples of appropriateconcentrations that may be used:

Interleukin 6 (IL-6): 50 to 150 ng/ml, one of about 50 ng/ml, 60 ng/ml,70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 110 ng/ml, 120 ng/ml, 130ng/ml, 140 ng/ml or 150 ng/ml;

Interleukin 7 (IL-7): 5 to 15 ng/ml, one of about 5 ng/ml, 6 ng/ml, 7ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14ng/ml or 15 ng/ml;

Interleukin 12 (IL-12): 5 to 15 ng/ml, one of about 5 ng/ml, 6 ng/ml, 7ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14ng/ml or 15 ng/ml;

Interleukin 15 (IL-15): 5 to 15 ng/ml, one of about 5 ng/ml, 6 ng/ml, 7ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14ng/ml or 15 ng/ml.

Table 1 below provides examples of certain embodiments of methods of thedisclosure.

TABLE 1 Examples of Elements of a Method Embodiments Examples forEmbodiments First Stimulation Source of T-cells Peripheral bloodmononuclear cells (PBMC) Non-adherent PBMC Antigen-presenting Dendriticcells (DC)s, PBMCs or B-blasts cells (APC) Cytokines Combinations ofIL-15 and IL-7, optionally with IL-6 and/or IL-12 and/or IL-21 and/orIL-4 Antigen Viral pepmixes for HPV Second stimulation Source of T-cellsProduct of first stimulation APCs DC PBMCs Autologous activated T-cells(AATC) Cytokines IL-15 and IL-7, preferably no IL-6 or IL-12 andoptionally IL-15 and IL-7 are the only interleukins Antigen Viralpepmixes for HPV Third stimulation Source of T-cells Product of secondstimulation (and subsequent stimulations as desired) APCs Pepmix-loadedAATCs + costimulatory cells Cytokines IL-15 and IL-7, preferably no IL-6or IL-12 and optionally IL-15 and IL-7 are the only interleukinsCostimulatory cells Cells expressing CD86, 4-1BB, and CD83, e.g., K562cells

Thus, in particular embodiments, a population of T-cells (wherein thepopulation may comprise substantially all T-cells or wherein thepopulation of T-cells is within another population of cells, such aswithin PBMCs) is exposed to a population of APCs to generate T celllines having particular characteristics, including at least: a)effectiveness at targeting HPV16 E6 and/or E7 and/or effectiveness attargeting HPV18 E6 and/or E7; b) polyclonality; c) TH1 bias; or d) acombination thereof. The generated T cell lines may be produced to beeffective at targeting HPV species 7 (HPV-18, -39, -45, -59, -68) andspecies 9 (HPV-16, -31, -33, -35, -52, -58, -67), and types 6 and 11,and this may be the results of pepmixes directed to any one or more ofthe following antigens: E1, E2, E3, E4, E5, E6, E7, L1, and/or L2.

In some cases, T-cells are stimulated more than once, and differentstimulation steps may or may not expose the population of cells to thesame conditions. In specific embodiments, a first stimulation hasconditions different from a subsequent stimulation, including a secondstimulation and/or a third stimulation. In specific embodiments, a firststimulation step of the method utilizes APCs that are pepmix-loaded DCsor pepmix-loaded PBMCs and utilizes IL-7 and IL-15. This stimulationstep may optionally be repeated one or more times.

In certain embodiments of the methods, between days 8 and 10 followingan initial exposure of the peripheral blood T-cells (or PBMCs) to thepepmix or APCs, there may be a re-stimulation of the PBMCs on day 8, day9, or day 10, but not later, and then a subsequent re-stimulation mayoccur on day 15, day 16, or day 17.

In a stimulation step that is subsequent to the first stimulation step(including optional repeats of the first stimulation step), theresultant T-cells obtained after the first stimulation (and which may bein a heterogeneous population of cells) are exposed to pepmix-loaded DCsor pepmix-loaded PBMCs and/or autologous activated T-cells. In astimulation that is subsequent to first and second stimulation steps,T-cells obtained after the second or later stimulation (and which mayreside in a heterogeneous population of cells) are exposed topepmix-autologous activated T-cells. Costimulatory cells that may beutilized in any stimulation step include at least cells that expressCD86, 4-1BB, CD83, CD40, OX40, and/or CD80. In specific cases, thecostimulatory cells may be K562 cells.

In some embodiments, during the steps of the method the cells in cultureare modified. In specific embodiments, the cells are modified to harbora polynucleotide that expresses a gene product that renders the cellseffective or more effective for a specific purpose or function, such aseffective or more effective for targeting a particular target and/orenhanced in function for T-cell-mediated cytotoxicity, and/or modifiedto resist tumor antigen-specific cellular immunity, for example.

In some embodiments, the cells are modified to express a certainnon-natural receptor that allows the T-cells to effectively or moreeffectively target a desired target cell, such as one that expresses acertain antigen. The cells may be modified to express a chimeric antigenreceptor (CAR), an αβ T-cell receptor, and so forth. The cells may bemodified to express an expression vector (that may be viral (includingretroviral, lentiviral, adenoviral, adeno-associated viral, and soforth) or non-viral) during the method at specific time points, such asthe vector being introduced between day 2 and 5 of culture, for example.In some embodiments the cells are exposed to the expression vectorwithin about 3 days after each stimulation, but in such cases themodification occurs in more differentiated T-cells that have less longterm potential (which in specific circumstances is desirable).

In specific embodiments, the cells are modified to express a CAR thattargets a cancer antigen, such as EphA2, HER2, GD2, Glypican-3, 5T4,8H9, a_(v)β₆integrin, B cell maturation antigen (BCMA) B7-H3, B7-H6,CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44,CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII,EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, fetal AchR,Folate Receptor α, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2,KDR, Lambda, Lewis-Y, MCSP, Mesothelin, Mucl, Muc16, NCAM, NKG2Dligands, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, Sp17, SURVIVIN, TAG72,TEM1, TEM8, VEGRR2, carcinoembryonic antigen, HMW-MAA, VEGF receptors,and/or other exemplary antigens that are present with in theextracellular matrix of tumors, such as oncofetal variants offibronectin, tenascin, or necrotic regions of tumors and othertumor-associated antigens or actionable mutations that are identifiedthrough genomic analysis and or differential expression studies oftumors, for example.

In some embodiments the cells are modified to resist tumorantigen-specific cellular immunity, e.g. mediated by transforming growthfactor beta (TGF-β). For example, the cells may be modified to express adominant negative receptor for TGF-beta (DNRII), e.g. as described inFoster et al., (Antitumor activity of EBV-specific T lymphocytestransduced with a dominant negative TGF-beta receptor. J Immunother.2008; 31:500-505, incorporated herein by reference). This may comprisetransfecting the cells with a retroviral expression vector encoding adominant negative TGF-β type II receptor (DNRII) modified by removal ofthe immunogenic hemagglutinin tag. Such modified T-cells have been shownto have a functional advantage over unmodified T-cells in the presenceof TGF-β-secreting tumor, including enhanced antitumor activity (Fosteret al., supra).

Methods according to the present invention may improve the rate ofexpansion for populations of virus-specific T-cells as compared to priorart methods. The rate of expansion for a T-cell population can beanalysed by methods well known to the skilled person. Methods includemeasuring the number of T-cells at one or more time points. For example,the number of T-cells can be determined after performing a methodaccording to the invention and compared to the number of T-cells at thebeginning of the method; fold expansion in the number of T-cells canthen be calculated.

Rates of expansion can also be determined by analysing cell division byT-cells over a period of time. Cell division for a given population ofT-cells can be analysed, for example, by in vitro analysis ofincorporation of ³H-thymidine or by CFSE dilution assay, e.g. asdescribed in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564,hereby incorporated by reference in entirety.

The improvement in the rate of expansion achieved by the methodsaccording to the present invention can be determined by performing amethod according to the invention, and comparing the expansion forT-cells in that method to a comparable, control method, e.g. as per themethod of Ramos et al., (J Immunother 2013; 36:66-76).

In some embodiments, the rate of expansion for a population of T-cellsin a method according to the present invention is one of at least 1.001times, 1.002 times, 1.003 times, 1.004 times, 1.005 times, 1.006 times,1.007 times, 1.008 times, 1.009 times, 1.01 times, 1.02 times, 1.03times, 1.04 times, 1.05 times, 1.06 times, 1.07 times, 1.08 times, 1.09times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times,1.7 times, 1.8 times, 1.9 times, or 2 times the rate of expansion in acomparable control method.

The rate of expansion may be of the virus-specific T-cell population, orthe total T-cell population.

The virus-specific T-cells generated/expanded according to the method ofthe present invention may at least retain the same functional propertiesas virus-specific T-cells generated/expanded according to prior artmethods. That is, the accelerated rate of expansion does not negativelyinfluence the functional properties of the expanded T-cells.

For example, in embodiments wherein the methods generate/expand apopulation of virus-specific T-cells, the T-cells display similarcytotoxicity to cells infected with or comprising/expressing a peptideof the virus as virus-specific T-cells expanded according to prior artmethods.

Cytotoxicity of expanded T-cells can be analysed e.g. by culturing theexpanded T-cell population with APCs presenting a peptide of the virusfor which the T-cell is specific at different effector (i.e. T-cell) totarget (i.e. APC) ratios, and measuring specific lysis of the APCs. Forexample, cytotoxicity of an HPV-specific CTL population can be analysedby measuring specific lysis of HPV-transformed LCL cells at differenteffector to target ratios.

B. Using Therapeutic T-Cells

In certain embodiments, cells produced by methods of the disclosure areprovided to an individual in need thereof for treatment of a medicalcondition, including one caused by a viral infection, or to target aviral infection in which no symptoms of a medical condition aredetectable or have manifested. As used herein “treatment” or “treating,”includes any beneficial or desirable effect on the symptoms or pathologyof a disease or pathological condition, and may include even minimalreductions in one or more measurable markers of the disease or conditionbeing treated, e.g., cancer. Treatment can involve optionally either thereduction or amelioration of symptoms of the disease or condition, orthe delaying of the progression of the disease or condition. “Treatment”does not necessarily indicate complete eradication or cure of thedisease or condition, or associated symptoms thereof.

In the methods encompassed by the disclosure, the therapeutic T-cellsare utilized to treat viral-associated disease caused directly orindirectly by a single non-HPV virus or are otherwise provided to anindividual that is seropositive for a single non-HPV virus. In othercases, the therapeutic T-cells are utilized to treat viral-associateddisease(s) caused directly or indirectly by more than one virus or areotherwise provided to an individual that is seropositive for more thanone virus. In the collection of therapeutic T-cells, each T-cell and itsprogeny has specificity for only one peptide in one antigen from onevirus, and upon production of the collection of therapeutic T-cells, oneexpands a population of T-cell clones that together havemulti-specificity, such as for multiple epitopes in each viral antigen,for example.

In at least some methods of the disclosure, a therapeutically effectiveamount of the CTLs generated thereby are administered to an individual,for example, an individual known to have or suspected of having orsusceptible to having HPV16 and/or HPV18-associated disease. In specificembodiments, the cells are administered by injection, such asintravenous, intramuscular, intradermal, subcutaneous, intraperitonealinjection, and so forth, for example. In some embodiments, the CTLs arefurther defined as polyclonal CD4+ and CD8+ CTLs. The PBMCs may beallogeneic to the individual or may be autologous to the individual.

In certain cases, neoplasms are treated with cells of the disclosure,and the neoplasm may be benign, malignant, or a premalignant lesion thatcan lead to cancer. Thus, an individual may be treated with cellsproduced by methods of the disclosure at the premalignant lesion stageand/or after the lesion becomes malignant. The individual may have earlyor late stage cancer, and the skilled artisan is aware that the methodsof producing the cells may be tailored for such different stages ofcancer, such as by utilizing peptides for the APCs that are fromantigens associated with early vs. late stage cancer. In specificembodiments, the cancer may be primary, metastatic, recurrent,refractory, and so forth.

In certain cases, premalignant lesions that can lead to cancers, such aspremalignant lesions of the cervix, vulva, vagina, penis, larynx,oropharynx anus, and other upper aerodigestive areas, for example, aretreated with cells produced by methods of the disclosure. Thus, anindividual may be treated with cells produced by methods of thedisclosure at the premalignant lesion stage and/or after the lesionbecomes malignant. HPV-associated medical conditions that may be treatedwith cells produced by methods of the disclosure include at leastdysplasias of the genital area(s), cervical intraepithelial neoplasia,vulvar intraepithelial neoplasia, penile intraepithelial neoplasia, analintraepithelial neoplasia, cervical cancer, anal cancer, vulvar cancer,vaginal cancer, penile cancer, genital cancers, oropharyngeal cancer,nasopharyngeal carcinoma, oral papillomas and other upper aerodigestivelesions.

In some cases, one can determine the serotype that is associated with acancer before administration of the cells, although in some cases theserotype is not determined. In specific embodiments, HPV16-specific orHPV18-specific cells have activity for tumors that are HPV16 orHPV18-positive, respectively, although in some cases there iscross-reactivity with different HPV serotypes. The ability tocross-react may or may not be known, and in certain cases, for example,an individual with HPV16 infection or HPV16-associated medical conditionis administered HPV18-specific T-cells, and vice versa. In such cases,an individual may be treated with cells specific for a serotype in whichit is unknown if the individual has that serotype, yet the cells stillare therapeutically effective because of cross-reactivity.

In cases wherein the APCs of the stimulation steps of the method areloaded with HPV16 and HPV18 pepmixes together, the outcome ofadministration of T-cells expanded through such APCs is determined bywhether the individual has been exposed to the virus in question. Forexample, if an individual is infected with HPV18 and not HPV16, onlyHPV18-specific T-cells will respond, and this is because the infectionwill initially have stimulated a T-cell response to HPV 18. ThoseT-cells will expand in the individual and then become memory T-cells andwould be at higher numbers than T-cells specific for HPV16 that havenever been activated, for example.

The individual being treated may be known to have cancer, suspected ofhaving cancer, or at risk for having cancer (such as personal or familyhistory; being sexual active, including sexually promiscuous; and/orhaving a genetic predisposition, including one or more specificmarkers). An individual being treated may have the presence of the HPVvirus but there are not yet any deleterious symptoms of a HPV-relatedmedical condition. The individual may have a benign or malignantneoplasm. The individual may have early or late stage cancer, and theskilled artisan is aware that the methods of producing the cells may betailored for such different stages of cancer, such as by utilizingpeptides for the APCs that are from antigens associated with early vs.late stage cancer. In specific embodiments, the HPV-associated diseaseis malignant cancer of the mouth or genital region. In specificembodiments, the cancer may be primary, metastatic, recurrent,refractory, and so forth. The individual may be infected with HPV16and/or HPV18 as a result of sexual acts of any kind or intimate physicalcontact of any kind.

Any stage of HPV infection may be treated with cells encompassed by thedisclosure. The individual with established HPV-associated cancer beingtreated with methods of the disclosure include Carcinoma in Situ (Stage0), Stage I, Stage II, Stage III, or Stage IV (which may be determinedby MRI, CT scan, PET scan, etc.). Additionally, individuals withpre-cancer lesions (dysplasia) may also be treated.

In some embodiments, one or more administrations of the cells producedby methods of the disclosure are provided to an individual in needthereof. The length of time between different administrations may be ofany suitable duration, including on the order of 1-7 days, 1-4 weeks,1-12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. Multipleinfusions within about a year may be employed, in some cases. In caseswherein more than one administration of cells are provided to theindividual, the antigen to which the cells are targeted may or may notbe the same antigen that was targeted with the cells utilized in earlieradministration(s). For example, in a first administration of cells, thecells may target HPV18 E6, whereas in another administration of cells,the cells target HPV18 E7, or vice versa. Additional administration(s)may be required in cancers that become refractory, for example.Additional stimulations may be employed in conjunction with one or moreother types of cancer treatments.

In some cases, an individual is optionally determined to have HPVinfection by any suitable means in the art. Because HPV cannot becultured in cell cultures, one may utilize HPV infection diagnosismethods such as DNA tests utilizing PCR, Southern blot hybridization,and/or in situ hybridization, and these methods may or may not be usedin conjunction with colposcopy; acetic acid test; biopsy; physicalexamination; and/or Pap smear, for example.

In specific embodiments, a male individual is provided an effectiveamount of cells produced by methods of the disclosure to target HPV withwhich he is infected, and in such a case the individual thereafter has areduced chance of infecting another, such as a female individual throughsexual activity. The male individual may or may not be determined to beinfected with HPV prior to exposure to the cells of the methods of thedisclosure. In some cases, if an individual is shown to be infected withan oncogenic HPV, it would be worth treating him with cells to eliminatehis risk. If the cells were effective, they would also reduce thechances of him transmitting the virus to his partner.

In specific embodiments, the individual is immunocompromised (which forexample, may be defined as an individual whose ability to fightinfectious disease or cancer with the immune system is compromised orentirely absent). In specific embodiments, the immunocompromisedindividual has had a stem cell transplant (including hematopoietic stemcell transplantation), has had an organ transplant and/or has receivedone or more cancer treatments, including chemotherapy or radiation, forexample. In some cases, the individual has acquired or inherited immunedeficiency disorder. In some embodiments, those that areimmunocompromised by their disease and/or its treatment are providedmethods and/or compositions of the disclosure.

Methods of medical treatment may involve treatment of cancer by a methodof ameliorating, treating, or preventing a malignancy in a human subjectwherein the steps of the method assist or boost the immune system ineradicating cancerous cells. Such methods may include the administrationof cells, according to the present invention that invoke an active (orachieve a passive) immune response to destroy cancerous cells. Methodsof treatment may optionally include the co-administration of biologicaladjuvants (e.g., interleukins, cytokines, Bacillus Comette-Guerin,monophosphoryl lipid A, etc.) in combination with conventional therapiesfor treating cancer such as chemotherapy, radiation, or surgery. Methodsof treatment may involve administering a composition according to thepresent invention as a vaccine that works by activating the immunesystem to prevent or destroy cancer cell growth. Methods of medicaltreatment may also involve in vivo, ex vivo, and adoptiveimmunotherapies, including those using autologous and/or heterologouscells or immortalized cell lines.

III. Pharmaceutical Compositions

In accordance with this disclosure, the term “pharmaceuticalcomposition” relates to a composition for administration to anindividual. In a particular embodiment, the pharmaceutical compositioncomprises a composition comprising therapeutic immune cells forparenteral, transdermal, intraluminal, intra-arterial, intrathecal orintravenous administration or for direct injection into a neoplasm, suchas a cancer. It is in particular envisaged that the pharmaceuticalcomposition is administered to the individual via infusion or injection.Administration of the suitable compositions may be effected by differentways, e.g., by intravenous, subcutaneous, intraperitoneal,intramuscular, topical or intradermal administration.

The pharmaceutical composition of the present disclosure may furthercomprise a pharmaceutically acceptable carrier. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions, etc.Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A particular dosage for administration mightbe in the range of 2×10⁷ cells per m² to 1×10¹⁰ cells per m² of bodysurface area. Progress can be monitored by periodic assessment.

The compositions of the disclosure may be administered locally orsystemically. In a preferred embodiment, the pharmaceutical compositionis administered subcutaneously and in an even more preferred embodimentintravenously. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishes,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. In addition, the pharmaceutical composition of thepresent disclosure might comprise proteinaceous carriers, like, e.g.,serum albumin or immunoglobulin, preferably of human origin. It isenvisaged that the pharmaceutical composition of the disclosure mightcomprise, in addition to the cells as described in this disclosure,further biologically active agents, depending on the intended use of thepharmaceutical composition.

IV. Combination Therapy

In certain embodiments of the disclosure that concern CTLs generatedagainst HPV antigen(s), methods of the present disclosure for clinicalaspects are combined with other agents effective in the treatment ofhyperproliferative disease, such as anti-cancer agents. An “anti-cancer”agent is capable of negatively affecting cancer in a subject, forexample, by killing cancer cells, inducing apoptosis in cancer cells,reducing the growth rate of cancer cells, reducing the incidence ornumber of metastases, reducing tumor size, inhibiting tumor growth,reducing the blood supply to a tumor or cancer cells, promoting animmune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions may beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This may be achieved by contacting the cancer cell with asingle composition or pharmacological formulation that includes bothagents, or by contacting the cancer cell with two distinct compositionsor formulations, at the same time, wherein one composition includes theexpression construct and the other includes the second agent(s). Inother cases, administration of the cells and a second composition may beseparate and may have separate administration routes and/or carriers.

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- and/orradiotherapy by combining it with additional therapy. In the context ofthe present disclosure, it is contemplated that cell therapy could beused similarly in conjunction with chemotherapeutic, radiotherapeutic,and/or immunotherapeutic intervention, for example.

Alternatively, the present inventive therapy may precede or follow theother agent treatment by intervals ranging from minutes to weeks,months, or years. In embodiments where the other agent and presentinvention are applied separately to the individual, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that the agent and inventive therapy would stillbe able to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It is expected that the treatment cycles would be repeated as necessary.It also is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the inventivecell therapy.

Examples of HPV-associated cancer treatments (as an example) that may beused in conjunction with cells produced from methods of the disclosureinclude at least the following: 1) surgery (tumor resection, neckdissection, conization, hysterectomy, and so forth.); 2) drug therapythat may include Avastin® (Bevacizumab); Blenoxane (Bleomycin);Hycamtin® (Topotecan Hydrochloride); or a combination thereof; 3)radiotherapy; 4) immunotherapy other than that of the disclosure; 5)hormone therapy; or 6) a combination thereof.

V. Kits of the Disclosure

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, a library of pepmixes may be comprised in a kit,any type of cells may be provided in the kit, and/or reagents formanipulation of pepmixes and/or cells may be provided in the kit.Cytokine(s) or means of producing them (such as vectors that encodethem) may be included in the kit. Cell culture reagents and/orapparatus(es) may be included. The component(s) are provided in suitablecontainer means.

In one embodiment a kit may comprise a container comprising a quantityof T-cells obtained by a method of the present invention formulated foradministration to a subject (e.g. by admixture with a suitable carrier,excipient, diluent, or adjuvant) preferably by infusion, more preferablyfor administration by infusion in a method of autologous adoptivecellular immunotherapy. The kit may be maintained at a predeterminedtemperature, e.g. less than about 4° C., less than about −2° C. or lessthan about −50° C. The kit may further comprise instructions for thestorage and/or transport of the kit and/or for the administration of theT-cells.

The kits may comprise a suitably aliquoted compositions of the presentinvention. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there are more than one componentin the kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the components in closeconfinement for commercial sale. Such containers may include injectionor blow molded plastic containers into which the desired vials areretained.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

In some cases, reagents and/or devices to detect HPV infection may beincluded in the kit. Examples include swabs, spatulas, cytobrushes,slides, cover slips, cytology sample collection receptacle(s), and soforth. Additional drugs for HPV infection or cancer may be included inthe kit, such as Bevacizumab; Bleomycin; Topotecan Hydrochloride; or acombination thereof.

VI. High IL-15

In some embodiments of aspects of the present disclosure stimulationsare performed in the presence of IL-15. In some embodiments stimulationsare performed in the presence of IL-7 and IL-15. In some embodimentsstimulations are performed in the presence of IL-7 and IL-15 only. Insome embodiments stimulations are performed in the presence of IL-7 andIL-15 and in the absence of IL-6 and/or IL-12).

In embodiments of the present disclosure where stimulations areperformed in the presence of IL-15, the IL-15 may be present in theculture at a final concentration greater than 15 ng/ml. Final IL-15concentrations greater than 15 ng/ml may be referred to herein as highIL-15. In some embodiments, the IL-15 may be present in the culture at afinal concentration of one of 20-1000 ng/ml, 20-900 ng/ml, 20-800 ng/ml,20-700 ng/ml, 20-600 ng/ml, 20-500 ng/ml, 30-500 ng/ml, 40-500 ng/ml,50-500 ng/ml, 50-400 ng/ml, 50-300 ng/ml, 50-200 ng/ml, 50-175 ng/ml,50-150 ng/ml, 75-150 ng/ml, 75-125 ng/ml, 80-120 ng/ml, 90-110 ng/ml or100 ng/ml. Stimulations using high IL-15 are contemplated in particularin connection with methods in which the populations of HPV-specificimmune cells are generated/expanded from within a population of cells(e.g. blood cells, immune cells or PBMCs) depleted of CD45RA+ cells.

Stimulations using high IL-15 may be provided with advantageousproperties. For example, methods comprising stimulations using highIL-15 may be useful to generate/expand populations of immune cellsspecific for HPV with improved efficiency (e.g. greater fold expansionwithin the same period of time). Methods comprising stimulations usinghigh IL-15 may be useful for generating/expanding populations of immunecells specific for HPV comprising a reduced number/frequency ofundesirable immune cell subsets (e.g. NK cells, regulatory T cells(Tregs) and/or naïve T cells), an increased proportion/frequency ofdesirable immune cell subsets (e.g. CD8+ T cells, CD8+ cytotoxic Tlymphocytes, CD4+ T cells, CD4+ T helper cells, IFNγ-producing cells,memory T cells, central memory T cells, antigen-experienced T cells,CD45RO+ T cells), and/or an increased proportion/frequency of virus-and/or antigen-specific cells in the generated/expanded population, ascompared to populations generated/expanded in accordance with prior artmethods.

VII. HLA-Negative LCLs

In aspects of the present disclosure the costimulatory cells used in astimulation step described herein may be cells of a lymphoblastoid cellline (LCL) lacking gene and/or protein expression of MHC class I and/orMHC class II. In some embodiment LCLs may lack surface expression of MHCclass I and MHC class II; such LCLs may be referred to herein as“HLA-negative LCLs”, “universal LCLs” or “ULCLs”.

LCLs can be prepared by viral transformation of B cells. LCLs aretypically produced by transformation of B cells with Epstein-Barr virus(EBV). Generation and characteristics of LCLs is described in detail,for example, in Hui-Yuen et al., J Vis Exp (2011) 57: 3321, and Hussainand Mulherkar, Int J Mol Cell Med (2012) 1(2): 75-87, both herebyincorporated by reference in their entirety. Briefly, LCLs can beproduced by incubation of PBMCs with concentrated cell culturesupernatant of cells producing EBV, for example B95-8 cells, in thepresence of cyclosporin A.

HLA-negative LCLs may lack surface expression of an MHC class Ipolypeptide and an MHC class II polypeptide. An “MHC class Ipolypeptide” refers to a constituent polypeptide of an MHC class Imolecule (i.e. a polypeptide complex of an MHC class I α chainpolypeptide and a B2M polypeptide). An “MHC class II polypeptide” refersto a constituent polypeptide of an MHC class II molecule (i.e. apolypeptide complex of an MHC class II α chain polypeptide and a MHCclass II β chain polypeptide). Surface expression refers to expressionof the relevant polypeptide/polypeptide complex which is detectable atthe cell surface (i.e. in or at the cell membrane). Surface expressioncan be analyzed e.g. on intact cells using an antigen-binding moleculespecific for a region of the polypeptide/polypeptide complex which isextracellular to the cell when the polypeptide/polypeptide complex isexpressed at the cell surface.

In some embodiments the HLA-negative LCLs display substantially nogene/protein expression of MHC class I and MHC class II, e.g. asdetermined by an appropriate method for detecting gene and/or proteinexpression. In some embodiments the HLA-negative LCLs displaysubstantially no surface expression of MHC class I and MHC class II,e.g. as determined by analysis by flow cytometry using an antibodycapable of binding to MHC class I and an antibody capable of binding toMHC class II. In such assays, the level of staining of the HLA-negativeLCLs by the relevant antibodies may not be significantly greater thanthe level of staining of the cells by appropriate negative controlantibodies of the same isotype.

HLA-negative LCLs may have been obtained by modification (e.g. to anucleic acid, e.g. by insertion, substitution or deletion of one or morenucleotides) to reduce/prevent gene and/or protein expression of one ormore polypeptides of an MHC class I molecule and an MHC class I molecule(e.g. B2M polypeptide, MHC class I α chain polypeptide (e.g. HLA-A,HLA-B or HLA-C), MHC class II α chain polypeptide (e.g. HLA-DPA1,HLA-DQA1, HLA-DQA2 or HLA-DRA) and/or MHC class II β chain polypeptide(e.g. HLA-DPB1, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB3, HLA-DRB4 orHLA-DRB5)). In some embodiments the HLA-negative LCLs comprisemodification to reduce/prevent gene and/or protein expression of an MHCclass I polypeptide (e.g. B2M) and modification to reduce/prevent geneand/or protein expression of one or more MHC class II polypeptides (e.g.HLA-DR, HLA-DQ, and HLA-DP) as compared to gene and/or proteinexpression by an unmodified LCL. In some embodiments the HLA-negativeLCLs comprise modification to reduce/prevent gene and/or proteinexpression of B2M, HLA-DRA, HLA-DQA1, HLA-DQA2, and HLA-DP. In someembodiments the HLA-negative LCLs may be obtained by targeted knockoutof genes encoding B2M, HLA-DRA, HLA-DQA1, HLA-DQA2, and HLA-DP, e.g.using sequence specific nucleases (SSNs); gene editing using SSNs isreviewed e.g. in Eid and Mahfouz, Exp Mol Med. 2016 October; 48(10):e265, which is hereby incorporated by reference in its entirety. In someembodiments modification to reduce/prevent gene and/or proteinexpression of an MHC class I polypeptide (e.g. B2M) and/or modificationto reduce/prevent gene and/or protein expression of one or more MHCclass II polypeptides (e.g. HLA-DR, HLA-DQ, and HLA-DP) is achievedusing CRISPR/Cas-9 systems comprising crRNA targeting nucleic acidencoding the relevant polypeptide(s). In some embodiments theHLA-negative LCLs are obtained by sequential knockout of genes encodingB2M, HLA-DRA, HLA-DQA1, HLA-DQA2, and HLA-DP. [0232] In some embodimentsthe HLA-negative LCLs additionally comprise modification to nucleic acidencoding one or more polypeptides necessary for EBVreplication/infection. LCLs comprising modification to reduce/preventEBV replication/infection may be described herein as being EBVreplication defective. Accordingly, in some embodiments the HLA-negativeLCLs are EBV replication defective. In some embodiments the HLA-negativeLCLs comprise modification to nucleic acid (e.g. by insertion,substitution or deletion of one or more nucleotides) encoding one ormore of BFLF1, BFLF2, BFRF1, BFRF2 and BFRF3 e.g. using SSNs. In someembodiments the HLA-negative LCLs comprise modification to nucleic acidencoding BFLF1 and/or nucleic acid encoding BFRF1. In some embodimentsmodification is achieved using CRISPR/Cas-9 systems comprising crRNAtargeting nucleic acid encoding the relevant polypeptide(s). In someembodiments the HLA-negative LCLs are obtained by a method comprisingculture in the presence of an agent suppressing viral replication (e.g.acyclovir). In some embodiments the EBV replication defectiveHLA-negative LCLs stimulate less proliferation of B cells from within apopulation of PBMCs following co-culture with the PBMCs as compared tothe level of proliferation of B cells from within a population of PBMCsfollowing co-culture of the PBMCs with LCLs described in the prior art.In some embodiments the EBV replication defective HLA-negative LCLs lackthe ability to promote outgrowth of B cells in a co-culture with PBMCs.HLA-negative LCLs modified to reduce/prevent gene and/or proteinexpression of one or more polypeptides necessary for EBV replication mayhave an improved safety profile as compared to LCLs lacking modificationto reduce/prevent gene and/or protein expression of one or morepolypeptides necessary for EBV replication.

The HLA-negative LCLs of the present disclosure are employed ascostimulatory cells in the manner in which costimulatory cells aretypically employed in methods for generating/expanding virus-specific Tcells. For example, the HLA-negative LCLs may be provided as anirradiated cell population, and at appropriate ratios to responder cellsand antigen presenting cells (APCs). In some embodiments theHLA-negative LCLs are irradiated or treated with a substance (e.g.mitomycin C) to prevent their proliferation, prior to use instimulations. Irradiation of LCLs in accordance with the present methodsis typically at 6000 to 12000 rads. In some embodiments the HLA-negativeLCLs are present in a stimulation comprising responder cells, APCs andHLA-negative LCLs at a ratio of responder cells:APCs:HLA-negative LCLsof one of 1:1:1 to 1:1:10, e.g. about 1:1:5.

HLA-negative LCLs described herein may be provided with advantageousproperties relevant to their use as costimulatory cells in methods forgenerating/expanding populations of HPV-specific immune cells. Forexample, the HLA-negative LCLs may be useful to generate/expandpopulations of immune cells specific for HPV with improved efficiency(e.g. greater fold expansion within the same period of time). TheHLA-negative LCLs may be useful in methods for generating/expandingpopulations of immune cells specific for HPV comprising a reducednumber/frequency of undesirable immune cell subsets (e.g. NK cells,regulatory T cells (Tregs) and/or naïve T cells), an increasedproportion/frequency of desirable immune cell subsets (e.g. CD8+ Tcells, CD8+ cytotoxic T lymphocytes, CD4+ T cells, CD4+ T helper cells,IFNγ-producing cells, memory T cells, central memory T cells,antigen-experienced T cells, CD45RO+ T cells), and/or an increasedproportion/frequency of virus- and/or antigen-specific cells in thegenerated/expanded population, as compared to populationsgenerated/expanded in accordance with prior art methods.

HLA-negative LCLs are useful for generating/expanding populations ofHPV-specific immune cells having reduced alloreactivity as compared toprior art methods for generating/expanding populations of HPV-specificimmune cells. Accordingly, HLA-negative LCLs are useful forgenerating/expanding populations of HPV-specific immune cells for use inboth autologous and allogeneic applications. In some embodiments theHLA-negative LCLs stimulate less proliferation of PBMCs in a coculturecomprising the HLA-negative LCLs and PBMCs obtained from an allogeneicdonor as compared to HLA-positive LCLs (i.e. LCLs described in the priorart). In some embodiments the HLA-negative LCLs stimulate lessproliferation of PBMCs in a coculture comprising the HLA-negative LCLsand PBMCs obtained from a HLA-matched donor as compared to HLA-positiveLCLs.

VIII. Dendritic Cells

In aspects of the present disclosure the dendritic cells used in astimulation step described herein may be derived from cells within apopulation of blood cells obtained from a subject. In some embodimentsthe dendritic cells are derived from cells within a population ofperipheral blood mononuclear cells (PBMCs). In some embodiments thedendritic cells are derived from CD14+ positive cells within apopulation of PBMCs. In some embodiments the dendritic cells aremonocyte-derived dendritic cells (also referred to herein as moDCs).

In some embodiments the dendritic cells are obtained by a processcomprising separating CD14+ cells from other cells (i.e. CD14− cells),e.g. from within a population of PBMCs. Separation may be performed byappropriate positive or negative selection. In some embodiments, CD14+positive cells may be positively selected from within a population ofcells using beads coated with antibody capable of binding specificallyto CD14. For example, CD14+ cells may be isolated by MACS cellseparation using CD14 MicroBeads (Miltenyi Biotec).

In some embodiments the dendritic cells are obtained by a processcomprising culturing cells in the presence of factors promotingdifferentiation of immature dendritic cells (also referred to herein asiDCs) from CD14+ cells. The cells cultured in the presence of factorspromoting differentiation of iDCs may be e.g. PBMCs, or CD14+ cellsisolated from a population of PBMCs. Cells may be cultured at anysuitable density, e.g. one of 0.1×10⁶ to 1×10⁶ cells/ml, 0.2×10⁶ to0.9×10⁶ cells/ml, 0.3×10⁶ to 0.8×10⁶ cells/ml, 0.4×10⁶ to 0.7×10⁶cells/ml or 0.5×10⁶ cells/ml. Factors promoting differentiation ofimmature dendritic cells may include IL-4 and/or GM-CSF. In someembodiments the culture comprises IL-4 and GM-CSF. The IL-4 may bepresent in the culture at a final concentration of one of 50-1000 IU/ml,100-800 IU/ml, 150-700 IU/ml, 200-600 IU/ml, 250-500 IU/ml, 300-500IU/ml or 400 IU/ml. The GM-CSF may be present in the culture at a finalconcentration of one of 100-1500 IU/ml, 200-1400 IU/ml, 300-1300 IU/ml,400-1200 IU/ml, 500-1100 IU/ml, 600-1000 IU/ml, 700-900 IU/ml or 800IU/ml. The culture may be for any suitable period of time fordifferentiation of immature DCs, e.g. one of 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

In some embodiments the dendritic cells used in a stimulation stepdescribed herein are obtained by a process comprising culturing cells(i.e. precursor cells of mature dendritic cells, e.g. immature dendriticcells) to mature dendritic cells. In some embodiments the processcomprises culturing the cells in the presence of factors promotingdifferentiation of mature DCs (e.g. moDCs). The cells cultured in thepresence of factors promoting differentiation of mature DCs may be e.g.PBMCs, CD14+ cells isolated (i.e. separated and/or purified) from apopulation of PBMCs, or immature dendritic cells (iDCs). Cells may becultured at any suitable density, e.g. one of 0.1×10⁶ to 1×10⁶ cells/ml,0.2×10⁶ to 0.9×10⁶ cells/ml, 0.3×10⁶ to 0.8×10⁶ cells/ml, 0.4×10⁶ to0.7×10⁶ cells/ml or 0.5×10⁶ cells/ml. Factors promoting differentiationof immature dendritic cells to mature DCs may be one or more of GM-CSF,IL-4, IL-1β, IL-6, TNFα, PGE-1, CD40L, Poly (I:C), MPLA, Resiquimod,IFNα, IFNγ or AmpB In some embodiments the culture comprises IL-4 andGM-CSF. In some embodiments the culture comprises GM-CSF, IL-4, IL-1β,IL-6, TNFα and PGE-1. In some embodiments the culture comprises GM-CSF,IL-4, IL-1β, IL-6 and TNFα. In some embodiments the culture comprisesGM-CSF, IL-4, IL-1β, IL-6, TNFα, PGE-1 and CD40L. In some embodimentsthe culture comprises GM-CSF, IL-4, IL-1β, IL-6, TNFα and CD40L. In someembodiments the culture comprises GM-CSF, IL-4, IL-1β, IL-6, TNFα, PGE-1and Poly (I:C). In some embodiments the culture comprises GM-CSF, IL-4,IL-1β, IL-6, TNFα and Poly (I:C). In some embodiments the culturecomprises GM-CSF, IL-4, IL-1β, IL-6, TNFα, PGE-1 and MPLA. In someembodiments the culture comprises GM-CSF, IL-4, IL-1β, IL-6, TNFα andMPLA. In some embodiments the culture comprises GM-CSF, IL-4, IL-1β,IL-6, TNFα, PGE-1 and Resiquimod. In some embodiments the culturecomprises GM-CSF, IL-4, IL-1β, IL-6, TNFα and Resiquimod. In someembodiments the culture comprises GM-CSF, IL-4, IL-1β, IL-6, TNFα, PGE-1and IFNα. In some embodiments the culture comprises GM-CSF, IL-4, IL-1β,IL-6, TNFα and IFNα. In some embodiments the culture comprises GM-CSF,IL-4, IL-1β, IL-6, TNFα, PGE-1 and IFNγ. In some embodiments the culturecomprises GM-CSF, IL-4, IL-1β, IL-6, TNFα and IFNγ. In some embodimentsthe culture comprises GM-CSF, IL-4, IL-1β, IL-6, TNFα, PGE-1, IFNα andIFNγ. In some embodiments the culture comprises GM-CSF, IL-4, IL-1β,IL-6, TNFα, IFNα and IFNγ. In some embodiments the culture comprisesGM-CSF, IL-4, IL-1β, IL-6, TNFα, PGE-1, Poly (I:C) and MPLA. In someembodiments the culture comprises GM-CSF, IL-4, IL-1β, IL-6, TNFα, Poly(I:C) and MPLA. In some embodiments the culture comprises GM-CSF, IL-4,MPLA and IFNγ. In some embodiments the culture comprises GM-CSF andIFNα. In some embodiments the culture comprises GM-CSF, IL-4, IFNγ andAmpB. The IL-4 may be present in the culture at a final concentration ofone of 50-1000 IU/ml, 100-800 IU/ml, 150-700 IU/ml, 200-600 IU/ml,250-500 IU/ml, 300-500 IU/ml or 400 IU/ml. The GM-CSF may be present inthe culture at a final concentration of one of 100-1500 IU/ml, 200-1400IU/ml, 300-1300 IU/ml, 400-1200 IU/ml, 500-1100 IU/ml, 600-1000 IU/ml,700-900 IU/ml or 800 IU/ml. The IL-1β may be present in the culture at afinal concentration of one of 1-100 pg/ml, 1-75 pg/ml, 5-50 pg/ml, 5-40pg/ml, 5-30 pg/ml, 5-25 pg/ml, 5-20 pg/ml, 5-15 pg/ml or 10 pg/ml. TheIL-6 may be present in the culture at a final concentration of one of10-1000 pg/ml, 10-750 pg/ml, 50-500 pg/ml, 50-400 pg/ml, 50-300 pg/ml,50-250 pg/ml, 50-200 pg/ml, 50-150 pg/ml or 100 pg/ml. The TNFα may bepresent in the culture at a final concentration of one of 1-100 pg/ml,1-75 pg/ml, 5-50 pg/ml, 5-40 pg/ml, 5-30 pg/ml, 5-25 pg/ml, 5-20 pg/ml,5-15 pg/ml or 10 pg/ml. The PGE-1 may be present in the culture at afinal concentration of one of 0.1-10 ng/ml, 0.1-7.5 ng/ml, 0.5-5 ng/ml,0.5-4 ng/ml, 0.5-3 ng/ml, 0.5-2.5 ng/ml, 0.5-2 ng/ml, 0.5-1.5 ng/ml or 1ng/ml. The CD40L may be present in the culture at a final concentrationof one of 10-1000 pg/ml, 10-750 pg/ml, 50-500 pg/ml, 50-400 pg/ml,50-300 pg/ml, 50-250 pg/ml, 50-200 pg/ml, 50-150 pg/ml or 100 pg/ml. ThePoly (I:C) may be present in the culture at a final concentration of oneof 0.1-10 μg/ml, 0.1-7.5 μg/ml, 0.5-5 μg/ml, 0.5-4 μg/ml, 0.5-3 μg/ml,0.5-2.5 μg/ml, 0.5-2 μg/ml, 0.5-1.5 μg/ml or 1 μg/ml. The MPLA may bepresent in the culture at a final concentration of one of 0.1-10 μg/ml,0.1-7.5 μg/ml, 0.5-5 μg/ml, 0.5-4 μg/ml, 0.5-3 μg/ml, 0.5-2.5 μg/ml,0.5-2 μg/ml, 0.5-1.5 μg/ml or 1 μg/ml. The Resiquimod may be present inthe culture at a final concentration of one of 0.1-10 μg/ml, 0.1-7.5μg/ml, 0.5-5 pg/ml, 0.5-4 μg/ml, 0.5-3 μg/ml, 0.5-2.5 μg/ml, 0.5-2μg/ml, 0.5-1.5 μg/ml or 1 μg/ml. The IFNα may be present in the cultureat a final concentration of one of 10-1000 ng/ml, 10-750 ng/ml, 50-500ng/ml, 50-400 ng/ml, 50-300 ng/ml, 50-250 ng/ml, 50-200 ng/ml, 50-150ng/ml or 100 ng/ml. The IFNγ may be present in the culture at a finalconcentration of one of 10-1000 ng/ml, 10-750 ng/ml, 50-500 ng/ml,50-400 ng/ml, 50-300 ng/ml, 50-250 ng/ml, 50-200 ng/ml, 50-150 ng/ml or100 ng/ml. The AmpB may be present in the culture at a finalconcentration of one of 0.1-10 μg/ml, 0.1-7.5 μg/ml, 0.5-5 μg/ml, 0.5-4μg/ml, 0.5-3 μg/ml, 0.5-2.5 μg/ml, 0.5-2 μg/ml, 0.5-1.5 μg/ml or 1μg/ml. The culture may be for any suitable period of time fordifferentiation of mature DCs, e.g. one of 6 hours to 5 days, 12 hoursto 4 days, 12 hours to 72 hours, 12 hours to 48 hours, 12 hours to 36hours, or 24 hours. In some embodiments the dendritic cells used instimulations are obtained by a process comprising culturing immature DCsin the presence of GM-CSF, IL-4, IL-1β, IL-6, TNFα and CD40L. In someembodiments the dendritic cells used in stimulations are obtained by aprocess comprising culturing immature DCs in the presence of GM-CSF,IL-4, MPLA and IFNγ.

Dendritic cells obtained by culture according to the methods describedherein may display higher surface expression of CD80 and/or CD83 ascompared to dendritic cells obtained by culture according to methodsdisclosed in the prior art. Populations of dendritic cells obtained byculture according to the methods described herein may comprise a higherproportion of cells displaying surface expression of CD80 and/or CD83 ascompared to populations of dendritic cells obtained by culture accordingto methods disclosed in the prior art.

Dendritic cells obtained by culture according to the methods describedherein may be provided with advantageous properties relevant to theiruse as antigen-presenting cells (APCs) in methods for expandingHPV-specific immune cells. For example, the DCs may be useful togenerate/expand populations of immune cells specific for HPV withimproved efficiency (e.g. greater fold expansion within the same periodof time). The DCs obtained by culture according to the methods describedherein may be useful in methods for generating/expanding populations ofimmune cells specific for HPV comprising a reduced number/frequency ofundesirable immune cell subsets (e.g. NK cells, regulatory T cells(Tregs) and/or naïve T cells), an increased proportion/frequency ofdesirable immune cell subsets (e.g. CD8+ T cells, CD8+ cytotoxic Tlymphocytes, CD4+ T cells, CD4+ T helper cells, IFNγ-producing cells,memory T cells, central memory T cells, antigen-experienced T cells,CD45RO+ T cells), and/or an increased proportion/frequency of virus-and/or antigen-specific cells in the generated/expanded population, ascompared to populations generated/expanded in accordance with prior artmethods.

VIII. CD45RA+ Cell Depletion

In aspects of the present disclosure, populations of HPV-specific immunecells are generated/expanded from a population of cells (e.g. bloodcells, immune cells or PBMCs) depleted of CD45RA+ cells (i.e. a CD45RA−cell population). In some embodiments the populations of HPV-specificimmune cells are generated/expanded from a population of PBMCs depletedof CD45RA+ cells. In some embodiments the depletion of CD45RA+ cells maybe achieved by separation of CD45RA− cells from other cells (i.e.CD45RA+ cells). Separation may be performed by appropriate positive ornegative selection. In some embodiments, CD45RA− cells may be separatedfrom CD45RA+ cells using beads coated with antibody capable of bindingspecifically to CD45RA. For example, CD45RA− cells may be separated fromCD45RA+ cells by MACS cell separation using CD45RA MicroBeads (MiltenyiBiotec). In some embodiments the population of cells may additionally bedepleted of CD14+ cells.

Depletion of CD45RA+ cells may remove NK cells, Tregs and/or naïve Tcells, preventing substantial outgrowth of these cell types duringstimulations according to the present disclosure. Depletion of CD45RA+cells may also enrich the initial population of cells for CD45RO+ cellsand/or antigen-experienced T cells, promoting preferential expansion ofthese desirable cell types in stimulations according to the presentdisclosure.

In some embodiments depletion of CD45RA+ cells is contemplatedespecially in methods employing IL-7 and IL-15 in one or morestimulations, and in particular where high concentrations of IL-15 areused (i.e. concentrations greater than 15 ng/ml, e.g. ˜100 ng/ml),because such methods may be particularly susceptible to the problem ofNK cell outgrowth. Depletion of CD45RA+ cells from the startingpopulation of cells used in expansions is also contemplated inparticular in methods employing K562cs costimulatory cells in methodsfor generating/expanding populations of HPV-specific immune cells,and/or methods in which HPV-specific immune cells are expanded frompopulations of immune cells (e.g. PBMCs) obtained from a patient, e.g. apatient having an HPV-associated disease.

Generation/expansion of populations of HPV-specific immune cells fromwithin a population of cells (e.g. blood cells, immune cells or PBMCs)depleted of CD45RA+ cells may be provided with advantages over prior artmethods for generating/expanding populations of HPV-specific immunecells. For example, the immune cell populations depleted of CD45RA+cells may be useful to generate/expand populations of immune cellsspecific for HPV with improved efficiency (e.g. greater fold expansionwithin the same period of time) Immune cell populations depleted ofCD45RA+ cells (e.g. PBMCs depleted of CD45RA+ cells) may be useful inmethods for generating/expanding populations of immune cells specificfor HPV comprising a reduced number/frequency of undesirable immune cellsubsets (e.g. NK cells, regulatory T cells (Tregs) and/or naïve Tcells), an increased proportion/frequency of desirable immune cellsubsets (e.g. CD8+ T cells, CD8+ cytotoxic T lymphocytes, CD4+ T cells,CD4+ T helper cells, IFNγ-producing cells, memory T cells, centralmemory T cells, antigen-experienced T cells, CD45RO+ T cells), and/or anincreased proportion/frequency of virus- and/or antigen-specific cellsin the generated/expanded population, as compared to populationsgenerated/expanded in accordance with prior art methods.

IX. Allogeneic and Autologous Applications

The methods disclosed herein are contemplated in the context ofgenerating/expanding HPV-specific immune cells for use in bothautologous and allogeneic applications, e.g. cellular immunotherapies.Populations of immune cells specific for HPV prepared in accordance withthe methods disclosed herein may be used after theirgeneration/expansion, or may be frozen for use at a later date.

In some embodiments the generated/expanded populations of immune cellsspecific for HPV are prepared for use in the subject from which theinitial population of immune cells from which the population isgenerated/expanded was obtained/derived. In some embodiments thegenerated/expanded populations of immune cells specific for HPV areprepared for use with any subject, e.g. a different subject to thesubject from which the initial population of immune cells from which thepopulation is generated/expanded was obtained/derived.

Accordingly, in some embodiments the generated/expanded populations ofimmune cells specific for HPV are adoptively transferred to the subjectfrom which the initial population of immune cells from which thepopulation is generated/expanded was obtained/derived. In someembodiments the generated/expanded populations of immune cells specificfor HPV are adoptively transferred to a different subject from thesubject from which the initial population of immune cells from which thepopulation is generated/expanded was obtained/derived.

EXAMPLES

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way,however, be construed as limiting the broad scope of the invention.

Example 1 Production of Therapeutic T-Cells

In some embodiments of the disclosure, there is a mechanism by which onecan rapidly generate a single preparation of T-cells, includingpolyclonal (for example, CD4+ and CD8+) CTLs, that are consistentlyspecific for a variety of antigens derived from one or more humanpapillomaviruses that can prove fatal. The disclosure is readilyadaptable to clinical implementation and can be used as an “off theshelf” HPV antiviral agent. The methods and compositions are readilyadaptable to clinical implementation and are useful as a safe andeffective HPV antiviral agent for individuals.

In specific embodiments, peripheral blood T-cells were stimulated withmonocyte-derived dendritic cells loaded with pepmixes [peptide librariesof 15-mers overlapping by 11 amino acids (aa)] spanning E6/E7, in thepresence or absence of specific accessory cytokines. The resultingT-cell lines were further expanded with pepmix-loaded activated B-cellblasts. There was successfully reactivation and expansion (>1200-fold)of E6-specific/E7-specific T-cells from 8/16 cervical and 33/52oropharyngeal cancer patients.

The presence of the cytokines interleukin (IL)-6, IL-7, IL-12, and IL-15is useful in the method, in specific embodiments of the methods. Theproduced T-cell lines possess the desirable characteristics ofpolyclonality, multiple T-cell subset representation (including thememory compartment) and a TH1 bias, and eliminate E6/E7 targets. Thedisclosure has shown that it is possible to robustly generate HPV16E6/E7-directed T-cell lines from patients with HPV16-associated cancers.Because the technique is scalable and good-manufacturingprocedures-compliant, these lines are useful for adoptive cellularimmunotherapy of patients with HPV16 cancers and may be applied to HPV18cancers also.

Known methods for producing T-cells for HPV16 are demonstrated in FIG.1A, showing results for 3 HPV-associated cancer patients (γIFN ELISpotassay obtained in cell lines obtained after stimulation of PBMCs by DCsloaded with only HPV16-pepmix). In FIG. 1B, results are shown for 2 ofthe patients whose results are also demonstrated in FIG. 1A, in additionto a third individual. FIG. 1B shows results of γIFN ELISpot assay forcell lines obtained after stimulation of PBMCs by DCs loaded withHPV16-pepmix and HPV18-pepmix. Reactivity against both HPV16 and HPV18antigens can be detected (not all patients will have reactivity againstboth serotypes).

Turning to specifics of the methods, in certain cases DCs are loadedwith HPV16-E6/E7 and HPV18-E6/E7 pepmix libraries. In such cases, thecell lines are able to recognize both HPV16 and HPV18 E6 and E7 antigens(instead of only HPV16 antigens, for example). In at least certaincases, expansion of the T-cells occurs in the presence of IL-7 and IL-15but not IL-2. The presence of IL-7 and IL-15 in conditions for themethod may or may not be at each step of stimulation and expansion. Insome embodiments, expansion of the HPV-specific T-cells after initialgeneration/expansion with DCs occurs not with autologous B-blasts loadedwith pepmixes in the presence of IL-15 but instead utilizes autologous,polyclonal activated T-cells loaded with pepmix, in the presence ofcostimulatory cells (CD80/CD86/CD83/4-1BBL), and IL-7 and IL-15.Employing these conditions, T-cell expansion occurs at a more rapidrate, at least 10-fold as that obtained by known methods, withsuccessful demonstration having occurred after 3 rounds of stimulationand without loss of specificity.

Summary of fold cellular expansion using the known method with 3HPV-associated cancer patients is provided in Table 2.

TABLE 2 Fold expansion at the end of each stimulation with known methodAfter 1^(st) stimulation After 2^(nd) stimulation After 3^(rd)stimulation Patient (with DC and IL- (with DC and IL- (with B-blasts andID 2/15) 2/15) IL-2/15) OPA 3.38 2.63 4.96 OPE 1.59 4.60 1.60 OPY 2.202.50 0.38

A summary of fold cellular expansion using a novel method of thedisclosure with 3 HPV-associated cancer patients is shown in Table 3.Fold expansion after 3 rounds of stimulation is on average approximately50 times higher than using a known method. Specificity is maintained(illustrated in #1).

TABLE 3 Fold expansion at the end of each stimulation with a method ofthe disclosure After 3^(rd) stimulation After 1^(st) stimulation After2^(nd) stimulation (with activated T- Patient (with DC and IL- (with DCand IL- cells, costim cells ID 7/15) 7/15) and IL-7/15) PDC 2.60 5.35151.20 PGD 5.00 6.36 120.00 PJK 3.58 5.53 60.00

Example 2 Protocol for the Expansion of HPV T-Cells

HPV stimulated T-cells (HPVST) are first activated by HPV E6/E7peptide-pulsed autologous dendritic cells (DCs) at 10-20:1 PBMC:DCratio, and cultured for 8 days in culture medium containing IL-6 (100ng/ml), IL-7 (10 ng/ml), IL-12 (10 ng/ml), IL-15 (10 ng/ml) (e.g. perthe first stimulation step described by Ramos et al., (J Immunother2013;36:66-76)).

A second stimulation step on day 9 is carried out using peptide-pulsedDCs at 5-10:1 PBMC:DC ratio in media containing IL-7 (10 ng/ml) andIL-15 (100 ng/ml).

Subsequent weekly stimulation/expansion steps are then carried toachieve a desired number of HPVSTs out using HPV E6/E7 peptide-pulsedautologous T-APC at 1:1 ratio, in the presence of equal number ofirradiated allogeneic K562-cs co-stimulatory cells, and in mediacontaining IL-7 (10 ng/ml) and IL-15 (100 ng/ml). The polyclonal T cells(T-APCs) are generated using a portion of the autologous PBMC isolatedfrom the venesected blood. The cells are activated by culturing in cellculture plates that are coated with anti-CD3 and anti-CD28 antibodies.The cells are then cultured to expand in the presence of IL-2 for 2weeks. The expanded T-APC can be cryopreserved for later use. 2-3 daysprior to using T-APC for HPVST re-stimulation (3rd cycle of HPVSTre-stimulation and onward), cryopreserved cells are thawed andre-stimulated in anti-CD3 and anti-CD28 antibody-coated cell cultureplates. On the day of HPVST re-stimulation, the T-APC cells areharvested and pulsed with the HPV E6/E7 peptides, followed by adding tothe on-going culture of HPVST at 1:1 ratio.

Example 3 In Vivo Expansion and Persistence of Infused HPVSTs in HumanPatients

HPVSTs obtained from Example 2 were transduced with a dominant negativereceptor for TGF-beta (DNRII) [see Foster et al., Antitumor activity ofEBV-specific T lymphocytes transduced with a dominant negative TGF-betareceptor. J Immunother. 2008; 31:500-505].

The HPVSTs were administered to two human patients and tested for invivo expansion and persistence. Patient #1 had widely metastaticoropharyngeal cancer and had discontinued prior therapy. Patient #2 hadoropharyngeal cancer metastatic to the neck and was receivingconcomitant nivolumab treatment, without prior response to nivolumabalone.

In vivo expansion and persistence of infused HPVSTs was assessed by qPCRfor the DNRII gene performed in PBMCs isolated from peripheral bloodfrom the patient. Data points in FIGS. 2 and 3 represent criticalpost-infusion intervals after the infusion of HPVSTs.

In patient #1 progressive expansion was observed (FIG. 2). In patient #2although expansion was limited (FIG. 3), with a peak at 6 weekscoinciding with re-infusion of HPVSTs, the patient had a partialclinical response. Six weeks after HPVST infusion patient #2 exhibited adecrease in disease burden measured by PET scan and physical examinationcompared to a pre-treatment baseline (FIG. 4).

Example 4 Optimization of DC Maturation

Blood samples were collected from EBV-reactive donors in heparinvacutainers and PBMCs were isolated from the blood samples by Ficolldensity gradient centrifugation. CD14 MicroBeads (Miltenyi Biotec) werethen used to isolate CD14+ cells from within the PBMCs by positiveselection. The number of cells was counted, and the CD14− cellpopulation was frozen and stored. The CD14+ cells were thendifferentiated to immature dendritic cells (iDCs). Briefly, CD14+ cellswere cultured in wells of a 24-well plate at a concentration of 0.5×10⁶cells/ml in DC cell culture medium containing 400 IU/ml IL-4 and 800IU/ml GM-CSF, for 5 days.

The iDCs were then matured to mature DCs by culture for 24 hours in DCcell culture medium according to the conditions shown in one of 1 to 20in Table 4 below:

TABLE 4 DC Maturation Culture Conditions Condition No. Conditions 1 800U/ml GM-CSF, 400 U/ml IL-4 2 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/mlIL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1 ng/ml PGE-1 3 800 U/ml GM-CSF,400 U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα 4 800 U/mlGM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1ng/ml PGE-1, 100 pg/ml CD40L 5 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/mlIL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 100 pg/ml CD40L 6 800 U/ml GM-CSF,400 U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1 ng/mlPGE-1, 1 μg/ml Poly (I:C) 7 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/mlIL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1 μg/ml Poly (I:C) 8 800 U/mlGM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1ng/ml PGE-1, 1 μg/ml MPLA 9 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/mlIL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1 μg/ml MPLA 10 800 U/ml GM-CSF,400 U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1 ng/mlPGE-1, 1 μg/ml Resiquimod 11 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/mlIL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1 μg/ml Resiquimod 12 800 U/mlGM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1ng/ml PGE-1, 100 ng/ml IFNα 13 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/mlIL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 100 ng/ml IFNα 14 800 U/ml GM-CSF,400 U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1 ng/mlPGE-1, 100 ng/ml IFNγ 15 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1β,100 pg/ml IL-6, 10 pg/ml TNFα, 100 ng/ml IFNγ 16 800 U/ml GM-CSF, 400U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1 ng/ml PGE-1,100 ng/ml IFNα, 100 ng/ml IFNγ 17 800 U/ml GM-CSF, 400 U/ml IL-4, 10pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 100 ng/ml IFNα, 100 ng/mlIFNγ 18 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6,10 pg/ml TNFα, 1 ng/ml PGE-1, 1 μg/ml Poly (I:C), 1 μg/ml MPLA 19 800U/ml GM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1β, 100 pg/ml IL-6, 10 pg/mlTNFα, 1 μg/ml Poly (I:C), 1 μg/ml MPLA 20 800 U/ml GM-CSF, 400 U/mlIL-4, 1 μg/ml MPLA, 100 ng/ml IFNγ

The mature DCs were then analyzed by flow cytometry for expression ofCD80, CD83, CCR7 and PD-L1. The previously frozen CD14− fraction wasthawed and rested in cell culture medium for 24 hours.

Immature and mature DCs were then pulsed with individual EBV antigenpepmixes (LMP1, LMP2, BRAF1 or EBNA1), and the peptide-pulsed DCs wereused to stimulate the autologous CD14− cells by coculture for 9 days inthe presence of IL-7 (10 ng/ml) and IL-15 (100 ng/ml). The expanded Tcell populations were then analyzed by flow cytometry for expression ofCCR7, CD45RO, EBV peptide reactivity and intracellular staining forIFNγ.

FIGS. 5A and 5B show CD83, CD80, CCR7 and PD-L1 expression bymonocyte-derived DCs derived from Donor 1 CD14+ PBMCs following cultureaccording to experimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11(see Table 4).

FIGS. 6A and 6B show CCR7 and CD45RO expression by CD4+ T cell and CD8+T cells obtained following stimulation of autologous CD14− PBMCs derivedfrom Donor 1 for 9 days with EBV peptide-pulsed mature DCs culturedaccording to experimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or11. No major differences in T cell memory phenotype were observed forthe expanded T cell populations.

FIG. 7 shows the total number of virus-specific T cells obtainedfollowing stimulation of autologous CD14− PBMCs derived from Donor 1 for9 days with EBV peptide-pulsed mature DCs cultured according toexperimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

FIG. 8 shows the proportions of IFNγ+CD8+ CTLs and IFNγ+CD4+ Th cellsobtained following stimulation of autologous CD14− PBMCs derived fromDonor 1 for 9 days with EBV peptide-pulsed mature DCs cultured accordingto experimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, asdetermined by ELISPOT analysis. Frequencies shown are subtracted forbackground signal obtained in control conditions performed in theabsence of EBV peptide stimulation.

FIG. 9 shows the proportions of EBV antigen-reactive IFNγ+CD8+ CTLs andIFNγ+CD4+ Th cells obtained following stimulation of autologous CD14−PBMCs derived from Donor 1 for 9 days with EBV peptide-pulsed mature DCscultured according to experimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11, as determined by ELISPOT analysis. Frequencies shown aresubtracted for background signal obtained in control conditionsperformed in the absence of EBV peptide stimulation. Donor 1 wasstrongly responsive to LMP2.

FIG. 10 shows the total number of virus-specific T cells and theproportion of IFNγ+CD8+ CTLs obtained following stimulation ofautologous CD14− PBMCs derived from Donor 1 for 9 days with EBVpeptide-pulsed mature DCs cultured under different experimentalconditions. Condition number 5 (‘CD40L wo’) was identified as apromising candidate for further evaluation because it was shown toperform better than condition 2 (‘Standard PGE’).

FIGS. 11A and 11B show CD83, CD80, CCR7 and PD-L1 expression bymonocyte-derived DCs derived from Donor 2 CD14+ PBMCs following cultureaccording to experimental conditions 1, 2, 3, 12, 13, 14, 15, 16, 17,18, 19 or 20.

FIGS. 12A and 12B show CCR7 and CD45RO expression by CD4+ T cell andCD8+ T cells obtained following stimulation of autologous CD14− PBMCsderived from Donor 2 for 9 days with EBV peptide-pulsed mature DCscultured according to experimental conditions 1, 2, 3, 12, 13, 14, 15,16, 17, 18, 19 or 20. No major differences in T cell memory phenotypewere observed for the expanded T cell populations.

FIG. 13 shows the total number of virus-specific T cells obtainedfollowing stimulation of autologous CD14− PBMCs derived from Donor 2 for9 days with EBV peptide-pulsed mature DCs cultured according toexperimental conditions 1, 2, 3, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

FIG. 14 shows the proportions of IFNγ+CD8+ CTLs and IFNγ+CD4+ Th cellsobtained following stimulation of autologous CD14− PBMCs derived fromDonor 2 for 9 days with EBV peptide-pulsed mature DCs cultured accordingto experimental conditions 1, 2, 3, 12, 13, 14, 15, 16, 17, 18, 19 or20, as determined by ELISPOT analysis. Frequencies shown are subtractedfor background signal obtained in control conditions performed in theabsence of EBV peptide stimulation.

FIG. 15 shows the proportions of EBV antigen-reactive IFNγ+CD8+ CTLs andIFNγ+CD4+ Th cells obtained following stimulation of autologous CD14−PBMCs derived from Donor 2 for 9 days with EBV peptide-pulsed mature DCscultured according to experimental conditions 1, 2, 3, 12, 13, 14, 15,16, 17, 18, 19 or 20, as determined by ELISPOT analysis. Frequenciesshown are subtracted for background signal obtained in controlconditions performed in the absence of EBV peptide stimulation. Donor 2was strongly responsive to LMP2.

FIG. 16 shows the total number of virus-specific T cells and theproportion of IFNγ+CD8+ CTLs obtained following stimulation ofautologous CD14− PBMCs derived from Donor 2 for 9 days with EBVpeptide-pulsed mature DCs cultured under different experimentalconditions. Condition numbers 18, 19 and 20 (‘PolyIC MPLA wo’, ‘PolyICMPLA PGE’ and ‘MPLA IFNγ’) were identified as promising candidates forfurther evaluation as they were shown to perform better than condition 2(‘Standard PGE’).

The inventors then undertook further investigation of the four mostpromising DC maturation conditions 5, 18, 19 and 20 (see Table 4),extending the characterization to cells derived from three differentdonors.

FIGS. 17A and 17B show representative CD83, CD80, CCR7 and PD-L1expression by monocyte-derived DCs derived from three different donorsfollowing culture according to the experimental conditions 1, 2, 5, 18,19 and 20. FIG. 17C shows the proportions of CD80+CD83− DCs, FIG. 17Dshows the proportions of CD80+CD83+ DCs and 17E shows the proportions ofPD-L1+ DCs.

FIG. 18 shows the total number of virus-specific T cells obtainedfollowing stimulation of autologous CD14− PBMCs derived from the threedifferent donors for 9 days with EBV peptide-pulsed mature DCs culturedaccording to the experimental conditions 2, 5, 18, 19 and 20. Mature DCsexpanded under the different conditions had similar overall levels ofexpansion.

FIG. 19 shows the proportions of IFNγ+CD8+ CTLs and IFNγ+CD4+ Th cellsobtained following stimulation of autologous CD14− PBMCs derived fromthe different donors for 9 days with EBV peptide-pulsed mature DCscultured according to the experimental conditions 2, 5, 18, 19 and 20,as determined by ELISPOT analysis. Frequencies shown are subtracted forbackground signal obtained in control conditions performed in theabsence of EBV peptide stimulation.

FIG. 20 shows the proportions of EBV antigen-reactive IFNγ+CD8+ CTLs andIFNγ+CD4+ Th cells obtained following stimulation of autologous CD14−PBMCs derived from the different donors for 9 days with EBVpeptide-pulsed mature DCs cultured according to the experimentalconditions 2, 5, 18, 19 and 20 as determined by ELISPOT analysis.Frequencies shown are subtracted for background signal obtained incontrol conditions performed in the absence of EBV peptide stimulation.

FIG. 21 shows the scaled total number of virus-specific T cells and thescaled frequency of IFNγ+ T cells obtained following stimulation ofautologous CD14− PBMCs derived from the three donors for 9 days with EBVpeptide-pulsed mature DCs cultured under different experimentalconditions.

Overall, the results suggested that the best condition for DC maturationis culture in the presence of GM-CSF, IL-4, IL-1β, IL-6, TNFα and CD40L.Another good condition is culture in the presence of GM-CSF, IL-4, MPLAand IFNγ.

Example 5 Further Optimization of DC Maturation

Blood samples are collected from EBV-reactive donors in heparinvacutainers and PBMCs are isolated from the blood samples by Ficolldensity gradient centrifugation. CD14+ microbeads are then used toisolate CD14+ cells from within the PBMCs by positive selection. Thenumber of cells is counted, and the CD14− cell population is frozen andstored.

The CD14+ cells are then differentiated to immature dendritic cells(iDCs). Briefly, CD14+ cells are cultured in wells of a 24-well plate ata concentration of 0.5×10⁶ cells/ml in DC cell culture medium containing400 IU/ml IL-4 and 800 IU/ml GM-CSF, for 5 days.

The iDCs are then matured to mature DCs by culture for 24 hours in DCcell culture medium according to the conditions shown in one of 1, 2, 21or 25 in Table 5 below:

TABLE 5 DC Maturation Culture Conditions Condition No. Conditions 1 800U/ml GM-CSF, 400 U/ml IL-4 2 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/mlIL-1β, 100 pg/ml IL-6, 10 pg/ml TNFα, 1 ng/ml PGE-1 21 800 U/ml GM-CSF,100 ng/ml IFNα 22 800 U/ml GM-CSF, 400 U/ml IL-4, 100 ng/ml IFNγ, 1μg/ml AmpB

The mature DCs are then analyzed by flow cytometry for expression ofCD80, CD83, CCR7 and PD-L1. The previously frozen CD14− fraction isthawed and rested in cell culture medium for 24 hours.

Immature and mature DCs are then pulsed with individual EBV antigenpepmixes (LMP1, LMP2, BRAF1 or EBNA1), and the peptide-pulsed DCs areused to stimulate the autologous CD14− cells by coculture for 3 days or9 days in the presence of IL-7 (10 ng/ml) and IL-15 (100 ng/ml). Theexpanded T cell populations are then analyzed by flow cytometry forexpression of CCR7, CD45RO, EBV peptide reactivity and intracellularstaining for IFNγ.

DCs matured according to conditions 21 and 22 are expected to beassociated with advantageous properties as compared to DCs maturedaccording to condition 2.

Example 6 Characterization of Virus-Specific T Cells Expanded byStimulation Culture in the Presence of Different Costimulatory Cells

The inventors next investigated the use of K562cs cells and HLA-negativeLCLs (also referred to herein as universal LCLs (ULCLs)) ascostimulatory cells in stimulations in methods for generating/expandingpopulations of virus-specific T cells. Briefly, PBMCs were harvested,and pulsed with EBV pepmixes corresponding to EBV antigens (EBNA-1,LMP-1, LMP-2 and BARF-1) in the presence of IL-7 (10 ng/ml) and IL-15(100 ng/ml) to stimulate the expansion of EBV-specific T cells. On day9, and every 7 days thereafter, restimulation steps were performedeither with (i) EBV pepmix-pulsed, irradiated autologous ATCs, in thepresence of IL-7 (10 ng/ml) and IL-15 (100 ng/ml), and in the presenceof irradiated K562-cs co-stimulatory cells, at a ratio of respondercells:ATCs:K562-cs of 1:1:5, or (ii) EBV pepmix-pulsed, irradiatedautologous ATCs, in the presence of IL-7 (10 ng/ml) and IL-15 (100ng/ml), and in the presence of irradiated ULCLs, at a ratio of respondercells:ATCs:ULCLs of 1:1:5.

LCLs lacking surface expression of HLA class I and HLA class II (i.e.HLA-negative LCLs) were obtained by targeted knockout of genes encodingHLA class I and HLA class II molecules. The HLA-negative LCLs werefurther modified to knockout genes necessary for EBV replication. Threedifferent ULCL clones (#4, #5 and #13) were analyzed.

FIGS. 22A and 22B show the results of two separate experiments,demonstrating that the overall fold expansion of the cells in culturewas higher for cells expanded using K562-cs cells as costimulatory cellsas compared to ULCLs. However, as shown in FIGS. 23A and 23B, ULCL clone#5 was found to expand greater numbers of virus-specific T cells, asdetermined by ELISPOT analysis of the number of IFNγ producing cells.

The inventors next analyzed the proportions of different cell typesexpanded after the indicated number of stimulations using K562-cs orULCLs as costimulatory cells, by flow cytometry using antibodiesspecific for cell surface markers. FIGS. 32A to 32C show scatterplotsfrom a representative donor (n=7) showing the proportions of CD3-CD56+NK cells, CD4+ T cells, CD8+ T cells, gamma delta T cells and alpha betaT cells in the expanded populations. Similar proportions of CD3-CD56+ NKcells were found in the expanded populations (FIG. 24A), whilstpopulations obtained by stimulations using K562cs cells tended to have areduced proportion of CD4+ T cells in the expanded populations (FIG.24B), and populations obtained by stimulations using ULCLs tended tohave an increased proportion of alpha beta T cells in the expandedpopulations (FIG. 24C). FIG. 25 shows representative results ofcharacterization of gamma delta TCR expression by ULCL clones #5 and#13, as compared to expression by K562cs cells.

The inventors next analyzed the frequency of antigen-specific T-cells inthe populations of cells expanded by stimulations using ULCL clone #5 or#13, or K562cs cells by ELISPOT analysis of IFNγ production at the endof the second and third stimulations. The results obtained from twodifferent donors are shown in FIGS. 26A and 26B. More virus-specificcells were detected where cells had been expanded by stimulation usingULCL cells as costimulatory cells. FIGS. 27A to 27D show the number ofcells per 100,000 cells specific for the indicated EBV antigens for fourdifferent donors after the indicated number of stimulations using K562cscells, ULCL clone #5 or LCLs as costimulatory cells, as determined byELISPOT analysis.

The inventors extended the analysis of virus-specific T cell expansionto four stimulations, and found that stimulations employing ULCLs ascostimulatory cells expanded populations containing higher proportionsof virus-specific cells as compared to stimulations performed usingK562cs cells (FIGS. 28A to 28D). ULCL clone#4 cells were found not todiffer significantly from cells of the parental ULCL clone in relationto ability to expand IFNγ producing virus-specific T cells (FIGS. 29Aand 29B).

In further experiments using PBMCs obtained from two different donors,stimulations using ULCL clone #5 yielded populations of cells comprisingIFNγ producing virus-specific T cells at a higher frequency as comparedto the frequency in populations expanded using K562cs cells instimulations (FIGS. 30A and 30B), with similar fold expansion (FIGS. 31Aand 31B). FIGS. 32 and 33 show that the proportions of CD3+ and CD56+cells in the expanded populations did not differ significantly inpopulations expanded using ULCL clone #5 as compared to populationsexpanded using K562cs cells, and FIGS. 34 and 35 show that populationsexpanded using K562cs cells expanded slightly greater proportion ofcentral memory cells.

FIGS. 36A and 36B show results from experiments obtained using PBMCsobtained from two further donors, demonstrating that stimulations usingULCL clone #5 yielded populations of cells comprising IFNγ producingvirus-specific T cells at a higher frequency as compared to thefrequency in populations expanded using K562cs cells in stimulations. Inthese experiments overall fold expansion was higher when K562cs cellswere used in stimulations (FIGS. 37A and 37B). FIGS. 38 and 39 show thatthe proportions of CD3+ and CD56+ cells in the expanded populations didnot differ significantly in populations expanded using ULCL clone #5 ascompared to populations expanded using K562cs cells, and FIGS. 40 and 41show that populations expanded using K562cs cells expanded slightlygreater proportion of central memory cells.

Example 7 Further Characterization of HPV-Specific T Cells Expanded byStimulation Culture in the Presence of Different Combinations ofCytokines and with Different Costimulatory Cells

HPV stimulated T-cells (HPVST) were first activated by HPV E6/E7peptide-pulsed autologous dendritic cells (DCs) at 10-20:1 PBMC:DCratio, and cultured for 8 days in culture medium containing IL-6 (100ng/ml), IL-7 (10 ng/ml), IL-12 (10 ng/ml), IL-15 (10 ng/ml) (e.g. perthe first stimulation step described by Ramos et al., (J Immunother2013; 36:66-76)).

Subsequent weekly restimulation steps were then carried out as shown inTable 6, four a total of four stimulations:

TABLE 6 Second and subsequent stimulations Condition Conditions K562cs7/15 HPV E6/E7 peptide-pulsed autologous T-APC at 1:1 ratio, in thepresence of equal number of irradiated allogeneic K562-cs co-stimulatory cells, in cell culture media containing IL-7 (10 ng/ml) andIL-15 (100 ng/ml). ULCL 7/15 HPV E6/E7 peptide-pulsed autologous T-APCat 1:1 ratio, in the presence of equal number of irradiated HLA-negativeLCLs, in cell culture media containing IL-7 (10 ng/ml) and IL-15 (100ng/ml). K562cs 6/7/12/15 HPV E6/E7 peptide-pulsed autologous T-APC at1:1 ratio, in the presence of equal number of irradiated allogeneicK562-cs co- stimulatory cells, in cell culture media containing IL-6(100 ng/ml), IL- 7 (10 ng/ml), IL-12 (10 ng/ml) and IL-15 (10 ng/ml).ULCL 6/7/12/15 HPV E6/E7 peptide-pulsed autologous T-APC at 1:1 ratio,in the presence of equal number of irradiated HLA-negative LCLs, in cellculture media containing IL-6 (100 ng/ml), IL-7 (10 ng/ml), IL-12 (10ng/ml) and IL-15 (10 ng/ml).

At the end of each stimulation the cells were counted, and analysed byflow cytometry to determine the proportions of different cell types inthe expanded populations, and analyzed by ELISPOT to determine thefrequency IFNγ producing, virus-specific cells in the expandedpopulations.

FIG. 42 shows that HPV-specific T cells were present at a higherfrequency in populations of cells expanded by methods employingstimulations in the presence of IL-6, IL-7, IL-12 and IL-15 as comparedto stimulations in the presence of IL-7 and IL-15 only. HPV-specific Tcells were also present at a higher frequency in populations of cellsexpanded by methods using ULCLs as costimulatory cells as compared tomethods using K562cs cells as costimulatory cells.

FIG. 43 shows the overall fold expansion of cells expanded according tothe different protocols. Methods comprising stimulation in the presenceof IL-7 and IL-15 only were found to expand more HPV-specific T cells ascompared to methods comprising stimulation in the presence of IL-6,IL-7, IL-12 and IL-15. Methods comprising stimulations using ULCLs ascostimulatory cells were shown to expand more HPV-specific T cells ascompared methods comprising stimulations using K562cs cells ascostimulatory cells.

FIG. 44 shows the expression of CD3 and CD56 by cells stimulatedaccording to the different protocols, after the indicated number ofstimulations. Cells expanded by stimulation in the presence of IL-7 andIL-15 only were shown to have a greater proportion of CD3+CD56− cells ascompared to cells expanded by stimulation in the presence of IL-6, IL-7,IL-12 and IL-15. Cells expanded by stimulations using HLA-negative LCLsas costimulatory cells were shown have a greater proportion of CD3+CD56−cells as compared to cells expanded by stimulations using K562cs ascostimulatory cells.

FIG. 45 shows the expression of CD4 and CD8 by cells stimulatedaccording to the different protocols, after the indicated number ofstimulations. Cells expanded by stimulation in the presence of IL-6,IL-7, IL-12 and IL-15 were shown to have a greater proportion of CD8+cells as compared to cells expanded by stimulation in the presence ofIL-7 and IL-15 only.

FIG. 46 shows the expression of CD45RO and CCR7 by cells stimulatedaccording to the different protocols, after the indicated number ofstimulations. Cells expanded by stimulation in the presence of IL-7 andIL-15 only were shown to have a greater proportion of central memory(i.e. CD45RO+CCR7+) cells as compared to cells expanded by stimulationin the presence of IL-6, IL-7, IL-12 and IL-15.

Example 8 Generation of Virus-Specific T Cells from PBMCs Depleted ofCD45RA+ Cells

The inventors investigated modifications to the methods for expandingvirus-specific T cells to increase the frequency of viral-specificantigen-specific T-cells and to reduce the frequency of NK cells inexpanded populations.

FIG. 47 provides a schematic representation of steps of the method usedto generate virus-specific T-cells in this Example. Briefly, on day 0PBMCs (which were either depleted of CD45RA+ cells, or which were notdepleted of CD45RA+ cells) were pulsed with viral peptides in thepresence of IL-7 (10 ng/ml) and IL-15 (100 ng/ml or 5 ng/ml), or in thepresence of IL-4 and IL-7. At day 9, the cells were re-stimulated usingpeptide-pulsed irradiated autologous antigen-presenting activatedT-cells (ATCs) in the presence of irradiated HLA-negative LCLs ascostimulatory cells, and in the presence of IL-7 (10 ng/ml) and IL-15(100 ng/ml or 5 ng/ml), or in the presence of IL-4 and IL-7. At day 16cells were harvested and analyzed.

FIG. 48 shows results of ELISPOT analysis showing that methods usingIL-7 and IL-15 in stimulations expanded virus-specific T cells at ahigher frequency as compared to methods using IL-4 and IL-7 instimulations.

Methods comprising stimulation in the presence of IL-7 and IL-15 werefound to be able to expand viral antigen-specific T cells from PBMCsobtained from lymphoma patients (FIG. 49). Using IL-15 instead of IL-4was found to increase the frequency of antigen-specific T-cells inpatient EBV-specific T cells.

Investigation of the optimal concentration of IL-15 to be used instimulations revealed that a higher dose of IL-15 (100 ng/ml) was bestfor increasing the frequency of virus-specific T cells (100 ng per mLcompared to standard dose of 5 ng per mL)—see FIG. 50. FIG. 51demonstrates that high doses of IL-15 increased the proportion ofcentral memory EBV-specific T cells in the expanded cell population.

The inventors next investigated how to minimize NK cell outgrowth inpopulations of expanded cells generated from healthy donor and lymphomapatient PBMCs. Preferential outgrowth of NK cells was found to be higherin methods using IL-15 in stimulations (NK cell populations appear to belarger in populations obtained by stimulations using IL-15 and IL-7). Toaddress this, conditions were developed to avoid excessive NK celloutgrowth. Depletion of CD45RA+ cells from the PBMCs prior tostimulations was investigated. CD45RA is a naïve T-cell marker that isalso expressed on natural T-regulatory cells and NK cells, so it wasreasoned that depletion of CD45RA+ cells would remove the NK cells fromthe starting PBMC population. Depletion of CD45RA+cells also removes Tregulatory cells that can inhibit the outgrowth of antigen-specificT-cells, especially in cancer patients, and also removes naïve cellsthat can grow as bystander cells and dilute the antigen-specificT-cells. Depletion can be achieved by any suitable method, e.g. usingmagnetic labeling and separation (for example, using Miltenyi® Bioteccolumns). Use of antibody to deplete cells using magnetic beads ornanobubbles may also occur.

The process used to generation of pepmix-activated EBV-specific T cellsfrom CD45RA-depleted PBMCs is illustrated schematically in FIG. 53. Asshown therein, whole PBMCs populations were depleted of CD45RA (orCD45RO for comparative purposes), and beginning day 0 or day 1 the firststimulation (S1) EBVpepmix was added to the depleted cells in thepresence of IL-7 and IL-15 to produce EBVSTs. At the end of S1 andbeginning of the second stimulation S2 (for example, between day 8 andday 10), the EBVSTs were exposed to sufficient amounts of EBV-Pepmixpulsed ATCs and sufficient amounts of costimulatory cells (such asK562cs cells) in the presence of IL-7 and IL-15, but in the absence ofIL-2, to produce the desired pepmix-activated EBVSTs. FIG. 54demonstrates the results that CD45RA depletion (CD45RA+ PBMC fromhealthy donors were depleted using Miltenyi® columns and GMP gradeCD45RA-conjugated beads) was found to decrease the frequency ofCD3-CD56+ NK cells in the expanded population, which was associated withincreased proliferation of EBVSTs (FIG. 55). FIG. 56 illustrates theenhanced fold expansion of EBVSTs following CD45RA depletion fromhealthy donors at the end of a second stimulation step. In addition, theCD45RA depletion was found to enhance antigen specificity of EBVSTs atthe end of a second stimulation (for example, day 16) (see FIGS. 57 and58, both showing data for healthy donors). The increased antigenspecificity of EBVSTs generated by expansion from CD45RA+ cell depletedPBMCs was found to be sustained after a third stimulation (FIG. 59).

The effects of CD45RA depletion was then characterized in virus-specificT cells obtained by expansion from PBMCs obtain from lymphoma patients,chosen either because their EBVSTs grown without depletion showed highfrequencies of NK cells or because they failed to grow or showantigen-specificity. FIG. 60 shows the total NK cell population at theend of a second stimulation step in five lymphoma patients,demonstrating that CD45RA depletion decreased NK cell populationoutgrowth in lymphoma patient EBVSTs, and that this depletion increasedthe frequency of the antigen-specific T-cells (illustrated by IFN-yrelease ELlspot assay at the end of a second stimulation; FIG. 61). Thisexperiment was performed in the absence of dendritic cells for the firststimulation (i.e. CD45RA+ cell depleted PBMCs were contacted directlywith EBVPepmix). Similar to the results observed in methods using PBMCsfrom healthy donors, CD45RA depletion was found to increase antigenspecificity in EBVSTs from lymphoma patients (FIG. 62). Proliferation ofthe lymphoma patients' EBVSTs is demonstrated in FIG. 63. Furthermore,CD45RA depletion enhanced cytolytic activity against pepmix-pulsedautologous activated T-cells (aATCs); percentage lysis was observed ateffector to target ratio of 20:1 (FIG. 64).

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of generating or expanding a populationof immune cells specific for human papillomavirus (HPV), comprising astep of stimulating a population of immune cells by culture in thepresence of antigen-presenting cells (APCs) presenting a peptide of aHPV antigen, in the presence of human leucocyte antigen (HLA)-negativelymphoblastoid cell line cells (LCLs).
 2. The method according to claim1, wherein the HLA-negative LCLs are Epstein-Barr virus (EBV)replication defective.
 3. The method according to claim 1, wherein theAPCs are activated T cells (ATCs), dendritic cells (DCs) or B-Blasts(BBs).
 4. The method according to claim 3, wherein the DCs are derivedfrom CD14+ cells isolated from a population of peripheral bloodmononuclear cells (PBMCs).
 5. The method according to claim 3, whereinthe DCs are derived from CD14+ cells by a method comprising culturingthe CD14+ cells in the presence of IL-4 and GM-CSF to produce immatureDCs (iDCs).
 6. The method according to claim 5, wherein the method bywhich the DCs are derived from CD14+ cells further comprises culturingthe iDCs to produce mature DCs by culture in the presence of: (a)GM-CSF, IL-4, IL-1β, IL-6, TNFα and CD40L; (b) GM-CSF, IL-4, MPLA andIFNγ; (c) GM-CSF, IFNα; or (d) GM-CSF, IL-4, AmpB and IFNγ.
 7. Themethod according to claim 3, wherein the DCs are derived from immatureDCs (iDCs) by a method comprising culture in the presence of: (a)GM-CSF, IL-4, IL-1β, IL-6, TNFα and CD40L; (b) GM-CSF, IL-4, MPLA andIFNγ; (c) GM-CSF, IFNα; or (d) GM-CSF, IL-4, AmpB and IFNγ.
 8. Themethod according to claim 1, wherein the population of immune cellsstimulated is depleted of CD45RA+ cells.
 9. The method according toclaim 1, wherein the stimulation is performed by culture in the presenceof IL-7 and IL-15, optionally wherein the IL-15 is present in theculture at a final concentration greater than 15 ng/ml.
 10. The methodaccording to claim 1, wherein the stimulation is performed by culture inthe presence of IL-4, IL-6, IL-7 and IL-15.
 11. The method according toclaim 1, wherein the APCs or DCs have been pulsed with one or morepeptides corresponding to one or more HPV antigens.
 12. The methodaccording to claim 11, wherein the one or more HPV antigens are selectedfrom E1, E2, E3, E4, E5, E6 and E7.
 13. The method according to claim11, wherein the one or more HPV antigens are antigens of HPV16, HPV18,HPV1, HPV2 and/or HPV3.
 14. The method according to claim 1, wherein thepopulation of immune cells stimulated is obtained from a priorstimulation of immune cells by culture in the presence of APCspresenting a peptide of a HPV antigen, optionally from within apopulation of PBMCs.
 15. The method according to claim 1, wherein themethod further comprises one or more further steps comprisingstimulating a population of immune cells by culture in the presence ofAPCs presenting a peptide of a HPV antigen.
 16. A method of treating orpreventing a HPV-associated disease in a subject, the method comprisingadministering to a subject a therapeutically or prophylacticallyeffective quantity of a population of immune cells specific for HPVgenerated or expanded by a method comprising a step of stimulating apopulation of immune cells by culture in the presence ofantigen-presenting cells (APCs) presenting a peptide of a HPV antigen,in the presence of HLA-negative LCLs.
 17. A method of treating orpreventing a HPV-associated disease in a subject, the method comprising:(a) generating or expanding a population of immune cells specific forHPV by a method comprising a step of stimulating a population of immunecells by culture in the presence of antigen-presenting cells (APCs)presenting a peptide of a HPV antigen, in the presence of HLA-negativeLCLs, and (b) administering a therapeutically or prophylacticallyeffective quantity of the population of immune cells specific for HPVobtained at step (a) to a subject.
 18. The method according to claim 17,wherein the HPV-associated disease is a cancer.
 19. The method accordingto claim 18 wherein the cancer is selected from cervical cancer, analcancer, vulvar cancer, vaginal cancer, penile cancer, oropharyngealcancer, nasopharyngeal carcinoma, laryngeal papillomatosis, laryngealcancer, head and neck cancer, or a dysplasia of any of site thereof. 20.The method according to claim 1, wherein the immune cells are peripheralblood mononuclear cells (PBMCs).
 21. The method according to claim 16,wherein the immune cells are peripheral blood mononuclear cells (PBMCs).22. The method according to claim 17, wherein the immune cells areperipheral blood mononuclear cells (PBMCs).